Ssx-2 peptide analogs

ABSTRACT

Some embodiments relate to analogs of peptides corresponding to class I MHC-restricted T cell epitopes and methods for their generation. These analogs can contain amino acid substitutions at residues that directly interact with MHC molecules, and can confer improved, modified or useful immunologic properties. Additionally classes of analogs, in which the various substitutions comprise the non-standard residues norleucine and/or norvaline, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/155,929, filed Jun. 17, 2005, which claims priority under 35 U.S.C.§119(e) to U.S. Provisional Application No. 60/581,001, filed on Jun.17, 2004, entitled SSX-2 PEPTIDE ANALOGS, and to U.S. ProvisionalApplication No. 60/580,962, filed on Jun. 17, 2004, entitled NY-ESOPEPTIDE ANALOGS; each of which is incorporated herein by reference inits entirety. This application is related to U.S. patent applicationSer. No. 11/156,253, filed Jun. 17, 2005, now U.S. Pat. No. 8,202,841,which claims priority under 35 U.S.C. §119(e) to U.S. ProvisionalApplication No. 60/581,001, filed on Jun. 17, 2004, entitled SSX-2PEPTIDE ANALOGS, and to U.S. Provisional Application No. 60/580,962,filed on Jun. 17, 2004, entitled NY-ESO PEPTIDE ANALOGS; each of whichis incorporated herein by reference in its entirety.

INCORPORATION OF INFORMATION PROVIDED IN ELECTRONIC FORMAT

The instant application is being filed along with a request to transferthe last filed Sequence Listing in electronic, computer readable formfrom parent application, U.S. patent application Ser. No. 11/155,929,which Sequence Listing was filed on Nov. 15, 2011, along with a PDF fileof the sequence listing with identical sequence information. Theinformation in the electronic and paper formats of the Sequence Listingis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

In certain embodiments, the invention disclosed herein relates toanalogs of peptides corresponding to class I MHC-restricted T cellepitopes and methods for their generation. These analogs can containamino acid substitutions at residues that directly interact with MHCmolecules, and can confer improved, modified or useful immunologicproperties. In particular, epitope analogs from the tumor-associatedantigens SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase, and melan-A areidentified. Additionally classes of analogs, in which the varioussubstitutions comprise the non-standard residues norleucine and/ornorvaline, are disclosed.

2. Description of the Related Art

The Major Histocompatibility Complex and T Cell Target Recognition

T lymphocytes (T cells) are antigen-specific immune cells that functionin response to specific antigen signals. B lymphocytes and theantibodies they produce are also antigen-specific entities. However,unlike B lymphocytes, T cells do not respond to antigens in a free orsoluble form. For a T cell to respond to an antigen, it requires theantigen to be bound to a presenting complex known as the majorhistocompatibility complex (MHC).

MHC proteins provide the means by which T cells differentiate native or“self” cells from foreign cells. MHC molecules are a category of immunereceptors that present potential peptide epitopes to be monitoredsubsequently by the T cells. There are two types of MHC, class I MHC andclass II MHC. CD4+ T cells interact with class II MHC proteins andpredominately have a helper phenotype while CD8+ T cells interact withclass I MHC proteins and predominately have a cytolytic phenotype, buteach of them can also exhibit regulatory, particularly suppressive,function. Both MHC are transmembrane proteins with a majority of theirstructure on the external surface of the cell. Additionally, bothclasses of MHC have a peptide binding cleft on their external portions.It is in this cleft that small fragments of proteins, native or foreign,are bound and presented to the extracellular environment.

Cells called antigen presenting cells (APCs) display antigens to T cellsusing the MHC. T cells can recognize an antigen, if it is presented onthe MHC. This requirement is called MHC restriction. If an antigen isnot displayed by a recognizable MHC, the T cell will not recognize andact on the antigen signal. T cells specific for the peptide bound to arecognizable MHC bind to these MHC-peptide complexes and proceed to thenext stages of the immune response.

SUMMARY OF THE INVENTION SSX-2₄₁₋₄₉ Analog Embodiments

Embodiments include analogs of the MHC class I-restricted T cell epitopeSSX-2₄₁₋₄₉, KASEKIFYV (SEQ ID NO. 1), polypeptides comprising theseanalogs that can be processed by pAPC to present the epitope analogs,and nucleic acids that express the analogs. The analogs can have similaror improved immunological properties compared to the wild-type epitope.

One embodiment relates to an isolated SSX-2 peptide having a sequencecomprising 1 or more amino acid substitutions of the sequence KASEKIFYV(SEQ ID NO:1), in an amount sufficient to elicit cytokine productionfrom a T cell line generated by immunization against an epitope with thesequence KASEKIFYV (SEQ ID NO:1). In one aspect, the amount sufficientcan be not more than 10 uM. In a further aspect, the amount is not morethan 3 uM. In a further is not more than 1 uM. In a further aspect, theamount can be not more than 0.3 uM. In one aspect, the substitutions caninclude a standard amino acid. In a further aspect, the amount can benot more than 0.3 uM. In one aspect, the substitutions can include astandard amino acid, for example Tyr, Val, Leu, Ala, Ile, Met, Trp, Phe,Asp, Asn, or Ser. In a further aspect, the substitutions can include anon-standard amino acid. In one aspect, the non-standard amino acid canbe, for example, Nle, Nva, Abu, or a D-stereoisomer of a standard aminoacid. In a further aspect, the substitutions can include a modifiedterminal amino acid. In one aspect, the modified terminal amino acid canbe an amidated C-terminal amino acid. In a further aspect at least oneof the substitutions can be the addition of an amino acid, wherein theaddition is a C-terminal addition. In a further aspect, the peptidefurther can include the substitution of conserved amino acids at anysite, but preferably at the P3, P5, or P7 sites which are not expresslyinvolved in any MHC interactions.

A further embodiment relates to an isolated peptide of 9 amino acids, P1to P9, which can include one amino acid at each site. For example, P1can be K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,β-(3-benzothienyl)-Ala, or D-Lys; P2 can be A, L, V, I, M, D-Ala, NaI-2,Abu, Aib, Nle, or Nva; P3 can be S; P4 can be E, Q, Nle, or Nva; P5 canbe K; P6 can be I, L, V, Nle, or Nva; P7 can be F; P8 can be Y, F,Phe(4-F); and PΩ (P-omega) at P9 can be V, I, A, Nva, MeVal, or Abu. Insome instances, the sequence is not KASEKIFYV (SEQ ID NO. 1).

A further embodiment relates to an isolated peptide of 9 amino acids, P1to P9, which can include one amino acid at each site. For example, P1can be K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,β-(3-benzothienyl)-Ala, or D-Lys; P2 can be V, L, M, Abu, Nle, or Nva;P3 can be S; P4 can be E, Q, Nle, or Nva; P5 can be K: P6 can be I, L,V, Nle, or Nva; P7 can be F; P8 can be Y, F, Phe(4-F); and PΩ at P9 canbe V, I, A, Nva, MeVal, Abu, or V—NH₂.

A further embodiment relates to an isolated peptide of 9 amino acids, P1to P9, which can include one amino acid at each site. For example, P1can be K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,β-(3-benzothienyl)-Ala, or D-Lys; P2 can be A, L, V, M, Abu, Nle, orNva; P3 can be S; P4 can be E, Q, Nle, or Nva; P5 can be K; P6 can be I,L, V, Nle, or Nva; P7 can be F; P8 can be Y, F, Phe(4-F); P9 can be V;and PΩ at P10 can be I or L.

A further embodiment relates to an isolated peptide of 9 amino acids, P1to P9, which can include one amino acid at each site. For example, P1can be K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,β-(3-benzothienyl)-Ala, or D-Lys; P2 can be V; P3 can be S; P4 can be E,Q, Nle, or Nva; P5 can be K: P6 can be I, L, V, Nle, or Nva; P7 can beF; P8 can be Y, F, Phe(4-F); P9 can be V; and PΩ at P10 can be I, L, V,or Nle.

A further embodiment relates to an isolated peptide of 9 amino acids, P1to P9, which can include one amino acid at each site. For example, P1can be K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,β-(3-benzothienyl)-Ala, or D-Lys; P2 can be L; P3 can be S; P4 can be E,Q, Nle, or Nva; P5 can be K: P6 can be I, L, V, Nle, or Nva; P7 can beF; P8 can be Y, F, Phe(4-F); P9 can be V; and PΩ at P10 can be I, L, V,Nle or Nva.

A further embodiment relates to an isolated peptide having the sequence:K{L, V, M, I, D-Ala, D-Val, NaI-2, Aib, Abu, Nle, or Nva}SEKIFYV (SEQ IDNO. 116); or {F, Phg, Y, Phe(4-F), Phe(4-NO₂), O-methyl-Tyr, orβ-(3-benzothienyl-Ala}ASEKIFYV (SEQ ID NO. 117); or {Y, F, or W}{V, M,or I}SEKIFYV (SEQ ID NO. 118); or {F or W}LSEKIFYV (SEQ ID NO. 119); orK{A, V, or L}SEKIFYI (SEQ ID NO. 120); or K{L or V}SEKIFYV-NH₂ (SEQ IDNO. 121); or FVSEKIFY{I, A, Nva, Abu, or MeVal} (SEQ ID NO. 122); orFVS{Q, Nle, Nva}KIFYV (SEQ ID NO. 123); or FVSEK{L, V, Nle, or Nva}FYV(SEQ ID NO. 124); or FVSEKIF{F, Phe(4-F)}V (SEQ ID NO. 125); orKVSEKIFYV{I, L} (SEQ ID NO. 126); or KVSEKIFYV{I, L, V, or Nle} (SEQ IDNO. 127); or KLSEKIFYV{L, V, Nle, or Nva} (SEQ ID NO. 128).

A further embodiment relates to an isolated peptide having the sequence:K{L, V, M, Abu, Nle, or Nva}SEKIFYV (SEQ ID NO. 129); or {F or Phg}ASEKIFYV (SEQ ID NO. 130); or YVSEKIFYV (SEQ ID NO. 131); or F{L, V, orI}SEKIFYV (SEQ ID NO. 132); or W{L or I}SEKIFYV (SEQ ID NO. 133); or K{Vor L}SEKIFYI (SEQ ID NO. 134); or FVSEKIFY{I or Nva} (SEQ ID NO. 135).

A further embodiment relates to an isolated peptide having the sequence:K{V or L}SEKIFYV (SEQ ID NO. 136); or {F or Y}ASEKIFYV (SEQ ID NO. 137);or FVSEKIFYI (SEQ ID NO. 138).

A further embodiment relates to a class I MHC/peptide complex whereinthe peptide has the sequence of any of the peptides in the embodimentsdescribed above and elsewhere herein. In one aspect, the complex can becross-reactive with a TCR that recognizes a class I MHC/SSX-2₄₁₋₄₉complex. In a further aspect, the complex can be an HLA-A2/SSX-2₄₁₋₄₉complex.

A further embodiment relates an immunogenic composition that can includeany of the peptide embodiments described above and elsewhere herein. Inone aspect the peptide can have, for example, the sequence: K{L, V, M,Abu, Nle, or Nva}SEKIFYV (SEQ ID NO. 129); or {F or Phg}ASEKIFYV (SEQ IDNO. 130); or YVSEKIFYV (SEQ ID NO. 131); or F{L, V, or I}SEKIFYV (SEQ IDNO. 132); or W{L or I}SEKIFYV (SEQ ID NO. 133); or K{V or L}SEKIFYI (SEQID NO. 134); or FVSEKIFY{I or Nva} (SEQ ID NO. 135), or K{V or L}SEKIFYV(SEQ ID NO. 136); or {F or Y}ASEKIFYV (SEQ ID NO. 137); or FVSEKIFYI(SEQ ID NO. 138).

Some further embodiments relate to analogs of the MHC class I-restrictedT cell epitope NY-ESO-1₁₅₇₋₁₆₅, SLLMWITQC (SEQ ID NO. 98), polypeptidesthat include these analogs that can be processed by pAPC to present theepitope analogs, and nucleic acids that express the analogs. The analogscan have similar or improved immunological properties compared to thewild-type epitope.

One embodiment relates to an isolated NY-ESO-1₁₅₇₋₁₆₅ peptide having asequence comprising 1 or more amino acid substitutions of the sequenceSLLMWITQC (SEQ ID NO. 98), in an amount sufficient to elicit cytokineproduction from a T cell line generated by immunization against anepitope with the sequence SLLMWITQC (SEQ ID NO. 98). For example, in oneaspect, the amount sufficient can be not more than 10 uM. In a furtheraspect, the amount can be not more than 3 uM. Also, in a further aspect,the amount can be not more than 1 uM. In a further aspect, the amount isnot more than 0.3 uM. In one aspect, the substitutions can include astandard amino acid. In a further aspect, the substitutions can includea non-standard amino acid. In one aspect, the non-standard amino acidcan be, for example, Tyr, Val, Leu, Ala, Ile, Met, Nle, Nva, Trp, Phe,Asp, Asn, Ser, Abu, and a D-stereoisomer of a standard amino acid. In afurther aspect, the substitutions can include a modified terminal aminoacid. In one aspect, the modified terminal amino acid can be an amidatedC-terminal amino acid. In a further aspect at least one of thesubstitutions can be the addition of an amino acid, wherein the additionis a C-terminal addition.

One embodiment relates to an isolated peptide having a sequence inwhich:

-   -   P1 is S, F, K, or W;    -   P2 is L, I, V, Nle, or Nva;    -   P3 is L;    -   P4 is M, L, or N;    -   P5 is W;    -   P6 is I, A, L, V, or N;    -   P7 is T;    -   P8 is Q, E, D, or T;    -   PΩ at P9 is C, V, I, L, A, Nva, Nle, V—NH₂, or L-NH₂; and    -   wherein the sequence is not SLLMWITQ{C, V, I, L, A} (SEQ ID NO.        99),    -   FVLMWITQA (SEQ ID NO. 100), or FILMWITQ{L, I} (SEQ ID NO. 101).

Another embodiment relates to an isolated peptide having a sequence inwhich:

-   -   P1 is Y;    -   P2 is L, V, I, Nle, or Nva;    -   P3 is L;    -   P4 is M, L, or N;    -   P5 is W;    -   P6 is I, A, L, V, or N;    -   P7 is T;    -   P8 is Q, E, D, or T;    -   PΩ at P9 is V, I, L, Nva, Nle, V, V—NH₂, or L-NH₂; and    -   wherein the sequence is not YVLMWITL (SEQ ID NO. 102) or        YLLMWIT{I, L} (SEQ ID NO. 103).

A further embodiment relates to an isolated decamer peptide having asequence {S,Y}LLMWITQ{C, V}{L, I, Nle} (SEQ ID NO. 104).

Yet another embodiment relates to an isolated peptide having a sequenceSILMWITQ{C, V, L, A} (SEQ ID NO. 105), YLLMWITQ{Nva, Nle} (SEQ ID NO.106), F{L, V}LMWITQ{V, L, I} (SEQ ID NO. 107), Y{I, Nva, Nle}LMWITQV(SEQ ID NO. 108), YLLLWITQV (SEQ ID NO. 109), or TVLMWITQV (SEQ ID NO.110).

A further embodiment relates to an isolated peptide having a sequence{S, F}VLMWITQV (SEQ ID NO. 111), SLMWITQNva (SEQ ID NO. 112), orSNvaLMWITQV (SEQ ID NO. 113).

Still another embodiment relates to an isolated peptide having asequence SNvaLMWITQV (SEQ ID NO. 113).

Some embodiments relate to an isolated peptide. The peptide can includeor consist essentially of a sequence in which:

-   -   P0 is X, XX, or XXX, wherein X specifies any amino acid or no        amino acid; and    -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is A, L, V, I, M, D-Ala, NaI-2, Abu, Aib, Nle, or Nva; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   PΩ at P9 is V, I, A, Nva, MeVal, Abu, or V—NH₂, or P9 is V, and        PΩ at    -   P10 is I, L, V, Nle or Nva; and    -   PΩ+1 is X, XX, or XXX, wherein X specifies any amino acid or no        amino acid; and    -   wherein the sequence is not KASEKIFYV (SEQ ID NO. 1).

The isolated peptide can include or consist essentially of the sequence:

(SEQ ID NO. 116) K{L, V, M, I, D-Ala, D-Val, Nal-2, Aib, Abu, Nle,or Nva}SEKIFYV; or (SEQ ID NO. 117) {F, Phg, Y, Phe(4-F), Phe(4-NO₂),O-methyl-Tyr, or β-(3-benzothienyl-Ala}ASEKIFYV; or (SEQ ID NO. 118){Y, F, or W}{V, M, or I}SEKIFYV; or (SEQ ID NO. 119) {F or W}LSEKIFYV;or (SEQ ID NO. 120) K{A, V, or L}SEKIFYI; or (SEQ ID NO. 121)K{L or V}SEKIFYV-NH₂; or (SEQ ID NO. 122)FVSEKIFY{I, A, Nva, Abu, or MeVal}; or (SEQ ID NO. 123)FVS{Q, Nle, Nva}KIFYV; or (SEQ ID NO. 124) FVSEK{L, V, Nle, or Nva}FYV;or (SEQ ID NO. 125) FVSEKIF{F, Phe(4-F)}V; or (SEQ ID NO. 126)KASEKIFYV{I, L,}; or (SEQ ID NO. 127) KVSEKIFYV{I, L, V, or Nle}; or(SEQ ID NO. 128) KLSEKIFYV{L, V, Nle, or Nva}.

The isolated peptide can include or consist essentially of the sequence:

(SEQ ID NO. 129) K{L, V, M, Abu, Nle, or Nva}SEKIFYV; or(SEQ ID NO. 130) {F or Phg}A SEKIFYV; or (SEQ ID NO. 131) YVSEKIFYV; or(SEQ ID NO. 132) F{L, V, or I}SEKIFYV; or (SEQ ID NO. 133)W{L or I}SEKIFYV; or (SEQ ID NO. 134) K{V or L}SEKIFYI; or(SEQ ID NO. 135) FVSEKIFY{I or Nva}.

Also, the isolated peptide can include or consist essentially of thesequence:

(SEQ ID NO. 136) K{V or L}SEKIFYV; or (SEQ ID NO. 137) {F or Y}ASEKIFYV;or (SEQ ID NO. 138) FVSEKIFYI; or KVSEKIFYV.

Further, the isolated peptide can include or consist essentially of thesequence KVSEKIFYV (SEQ ID NO. 3).

The isolated peptide can have affinity for a class I MHC peptide bindingcleft. The MHC can be, for example, HLA-A2.

Some embodiments relate to a class I MHC/peptide complex wherein thepeptide can have the sequence of the peptide of claim 1. The class IMHC/peptide complex can be cross-reactive with a TCR that recognizes aclass I MHC/SSX-2₄₁₋₄₉ complex. The class I MHC/peptide complex can bean HLA-A2/SSX-2₄₁₋₄₉ complex.

Other embodiments relate to a polypeptide that includes a polypeptide asdescribed above and elsewhere herein, in association with a liberationsequence.

Still further embodiments relate to immunogenic compositions thatinclude a peptide as described above or elsewhere herein.

Others relate to nucleic acids encoding or nucleic acid means forexpressing a polypeptide as described above or elsewhere herein. Also,some relate to immunogenic compositions that include such nucleic acidsor nucleic acid means.

Some embodiments relate to methods of inducing, maintaining, oramplifying a CTL response. The methods can include intranodaladministration of a composition as described above and elsewhere herein.

Other embodiments relate to methods of entraining a class IMHC-restricted T cell response, which methods can include intranodaladministration a composition as described above or elsewhere herein plusan immunopotentiating agent.

Further embodiments relate to methods of inducing, maintaining, orentraining a CTL response, which methods can include intranodaladministration of a composition as described above and elsewhere herein.

Some embodiments relate to isolated peptides that include 1 to 3substitutions in the sequence KASEKIFYV (SEQ ID NO. 1) having anaffinity for a class I MHC binding cleft that is similar to or greaterthan the affinity of KASEKIFYV (SEQ ID NO. 1) for said class I MHCbinding cleft. The halftime of dissociation can be similar to or greaterthan the halftime of dissociation of KASEKIFYV (SEQ ID NO. 1) from saidclass I MHC binding cleft. The isolated peptide can be recognized by Tcells with specificity for the peptide KASEKIFYV (SEQ ID NO. 1).

Still further embodiments relate to isolated peptides that include orconsisting essentially of a sequence in which:

-   -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is A, L, V, I, M, D-Ala, NaI-2, Abu, Aib, Nle, or Nva; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   PΩ at P9 is V, I, A, Nva, MeVal, or Abu;    -   wherein the sequence is not KASEKIFYV (SEQ ID NO. 1);    -   or    -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is V, L, M, Abu, Nle, or Nva; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   PΩ at P9 is V, I, A, Nva, MeVal, Abu, or V—NH₂;    -   or    -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is A, L, V, M, Abu, Nle, or Nva; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   P9 is V; and    -   PΩ at P10 is I or L;    -   or    -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is V; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   P9 is V; and    -   PΩ at P10 is I, L, V, or Nle;    -   or    -   P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO₂), MeTyr,        β-(3-benzothienyl)-Ala, or D-Lys; and    -   P2 is L; and    -   P3 is S; and    -   P4 is E, Q, Nle, or Nva; and    -   P5 is K: and    -   P6 is I, L, V, Nle, or Nva; and    -   P7 is F; and    -   P8 is Y, F, Phe(4-F); and    -   P9 is V; and    -   PΩ at P10 is I, L, V, Nle or Nva.

Some embodiments relate isolated peptides that include or consistessentially of a sequence in which:

-   -   P0 is X, XX or XXX, wherein X specifies any amino acid or no        amino acid; and    -   P1 is S, F, K, W or Y; and    -   P2 is L, I, V, Nle, or Nva; and    -   P3 is L; and    -   P4 is M, L, or N; and    -   P5 is W; and    -   P6 is I, A, L, V, or N; and    -   P7 is T; and    -   P8 is Q, E, D, or T; and    -   PΩ at P9 is C, V, I, L, A, Nva, Nle, V—NH₂, or L-NH₂; and    -   PΩ+1 is X, XX, XXX, wherein X specifies any amino acid or no        amino acid; and    -   wherein the sequence is not SLLMWITQ{C, V, I, L, A} (SEQ ID NO.        99), FVLMWITQA (SEQ ID NO. 100), FILMWITQ{L, I} (SEQ ID NO.        101), YVLMWITL (SEQ ID NO. 102) or YLLMWIT{I, L} (SEQ ID NO.        103).    -   P1 is S, F, K, or W;    -   P2 is L, I, V, Nle, or Nva;    -   P3 is L;    -   P4 is M, L, or N;    -   P5 is W;    -   P6 is I, A, L, V, or N;    -   P7 is T;    -   P8 is Q, E, D, or T;    -   PΩ at P9 is C, V, I, L, A, Nva, Nle, V—NH₂, or L-NH₂; and    -   wherein the sequence is not SLLMWITQ{C, V, I, L, A} (SEQ ID NO.        99), FVLMWITQA (SEQ ID NO. 100), or FILMWITQ{L, I} (SEQ ID NO.        101);    -   or    -   P1 is Y;    -   P2 is L, V, I, Nle, or Nva;    -   P3 is L;    -   P4 is M, L, or N;    -   P5 is W;    -   P6 is I, A, L, V, or N;    -   P7 is T;    -   P8 is Q, E, D, or T;    -   PΩ at P9 is V, I, L, Nva, Nle, V, V—NH₂, or L-NH₂; and    -   wherein the sequence is not YVLMWITL (SEQ ID NO. 102) or        YLLMWIT{I, L} (SEQ ID NO. 103).

A further embodiment relates to a class I MHC/peptide complex whereinthe peptide can have the sequence of any of the peptides in theembodiments described above or elsewhere herein. In one aspect, thecomplex can be cross-reactive with a TCR that recognizes a class IMHC/NY-ESO-1₁₅₇₋₁₆₅ complex. In a further aspect, the complex can be anHLA-A2/NY-ESO-1₁₅₇₋₁₆₅ complex.

In one aspect of the above embodiments, the peptide can have affinityfor a class I MHC peptide binding cleft, such as HLA-A2.

A further embodiment relates to a polypeptide comprising the peptidesequence of any of the embodiments in association with a liberationsequence.

A further embodiment relates to an immunogenic composition that includesany of the peptide embodiments. In one aspect the peptide can have asequence as set forth herein.

A further embodiment relates to a nucleic acid encoding any of thepeptide embodiments, but preferably those which do not have non-standardamino acid substitutions. In a further aspect, the nucleic acid can beencoded in a vector.

A further embodiment relates to an immunogenic composition that includesthe nucleic acid encoding any of the peptide embodiments.

A further embodiment relates to a method of inducing a CTL response byintranodal administration of any of the compositions or peptides of theembodiments. In a further aspect, the method can allow for maintaining aCTL. In a further aspect, the method can allow for amplifying a class IMHC-restricted T cell response. In a further aspect, the method canallow for entraining a class I MHC-restricted T cell response. In afurther aspect, the method also can include an immunopotentiating agent.

Some embodiments relate to isolated peptides having a sequencecomprising 1 to 3 or 4 amino acid substitutions in a native epitopesequence, wherein a concentration of the peptide required to elicitcytokine production from a T cell line generated by immunization againstan epitope with the sequence is not more than a particularconcentration, for example, 10 uM, 1 uM, 0.3 uM, and the like. Thesubstitutions can include a standard amino acid, a non-standard aminoacid, and the like. The non-standard amino acid can be any of thosedescribed herein, for example, a D-stereoisomer of a standard aminoacid, Nva, or Nle. The substitutions can include a modified terminalamino acid, and the modified terminal amino acid can be an amidatedC-terminal amino acid. One of the substitutions can be the addition ofan amino acid, for example, the addition can be a C-terminal addition.

Other embodiments relate to peptides having an amino acid sequence thatincludes at least one difference from a sequence of a segment of atarget-associated antigen, the segment having known or predictedaffinity for the peptide binding cleft of a MHC protein, wherein the atleast one difference can be a Nle or Nva residue replacing a residue atan MHC-binding motif anchor position in said segment. The anchorposition can be a primary anchor position, for example, P2 or PΩ. Theanchor position can be an auxiliary anchor position. The difference caninclude a Nle or Nva residue replacing a hydrophobic residue in saidsegment. In some aspects I, L, or V can be a preferred residue in theMHC-binding motif anchor position. In some aspects the peptide can havea length of about 8 to about 14 amino acids or more preferably a lengthof 9 to 10 amino acids, for example.

The protein can be a human MHC protein, for example, class I MHCprotein. The MHC protein can be, for example, a type such as HLA-A2, A3,A24, A30, A66, A68, A69, B7, B8, B15, B27, B35, B37, B38, B39, B40, B48,B51, B52, B53, B 60, B61, B62, B63, B67, B70, B71, B75, B77, C4, Cw1,Cw3, Cw4, Cw6, Cw7, and Cw10. In some aspects, the MHC protein can beHLA-A2 or A24. The MHC can have an anchor residue binding pocket,wherein the pocket is homologous to the B- or F-pocket of HLA-A*0201.The MHC residues responsible for forming binding pockets, and whichbinding pockets accommodate epitope anchor residues and thus define thebinding specificity of the MHC molecule, are well understood in the art.One compilation of such information is found at the FIMM (FunctionalImmunology) web site at the hypertext transfer protocol (http://)“sdmc.lit.org.sg:8080/fimm/.” See also Schönbach C., Koh J. L. Y., ShengX., Wong L., and V. Brusic. FIMM, a database of functional molecularimmunology. Nucleic Acids Research, 2000, Vol. 28, No. 1 222-224; andSchönbach C., Koh J L, Flower D R, Wong L., and Brusic V. FIMM, adatabase of functional molecular immunology; update 2002. Nucleic AcidsResearch, 2002, Vol. 30, No. 1 226-229; each of which is herebyincorporated by reference in its entirety. Also, the peptide can have atleast one binding characteristic that is substantially the same as, orbetter than, a corresponding characteristic of said segment for saidMHC. For example, the binding characteristic can be elevated comparedwith that of said segment. Also, the binding characteristic can beaffinity or stability of binding for example.

The peptide can have an immunogenicity that is substantially the sameas, or better than, the immunogenicity of the segment. Theimmunogenicity can be increased. The immunogenicity can evoke an immuneresponse that is cross-reactive to said segment or can evoke a CTLresponse. The immunogenicity can be assessed, for example, using anMHC-tetramer assay, a cytokine assay, a cytotoxicity assay, by measuringan immune response recognizing the peptide, by measuring an immuneresponse recognizing said segment, using an in vitro immunizationssystem, or any other suitable method. The immunization system caninclude human cells. The immunogenicity can be assessed using an in vivoimmunization system, for example, one that includes a transgenic mouse.The peptide can have an at least similar binding characteristic as saidsegment for said MHC. For example, in some aspects what is considered tobe “similar” can be determined based upon the instant disclosure. Insome particular aspects “similarity” can be based upon, for example,peptide concentration for half-maximal binding, relative affinity,stability (half time of dissociation) and cross-reactivity/functionalavidity. As an example, a peptide can be considered similar if it hasresults or characteristics that are within twofold, even threefold,four, five or 10 fold of the value for the native peptide. Also, as anexample, for cross-reactivity/functional avidity a similar result can beone where the data are within three and 10-fold of the native peptide.As another example, percentage of binding values can be consideredsimilar when within 2, 3, 4, 5, 6, 7, 10, 15 or 20% of the nativepeptide. Also, ED50 values can be considered similar in some aspectswhen within 2- or 3-fold of native sequence. Similar halftime ofdissociation can be for example within 2- or 3-fold. As still anotherexample, for cross-reactivity a value that is about 2-fold differentfrom wild-type can be considered similar. These similar values areexemplary only and given in the context of some aspects of someembodiments. Other “similar” values can be determined based upon theother experiments and teachings herein.

The peptides can be immunologically cross-reactive with the segment. Thecross-reactivity can be assessed by immunizing with the segment andassaying recognition of the peptide. The cross-reactivity can beassessed by immunizing with the peptide and assaying recognition of thesegment.

The peptide as described above and elsewhere herein can be modified toinclude two differences, for example. In some instances each differenceindependently can include a Nle or Nva residue. In some instances onedifference can not include a Nle or Nva residue. Also, the peptide asdescribed above and elsewhere herein can include three or moredifferences.

The target-associated antigen can be a tumor-associated antigen. Thetarget-associated antigen can be a pathogen-associated antigen.

Other embodiments relate to immunogenic composition that include theinstant peptides as described above and elsewhere herein. Furtherembodiments relate to methods of immunization that include administeringsuch compositions to a mammal, for example, administering directly tothe lymphatic system.

Still other embodiments relate to methods of making a T cell epitopeanalogue. The methods can include providing an amino acid sequence of asegment of a target-associated antigen, the segment can have known orpredicted affinity for the peptide binding cleft of a MHC protein;changing at least one amino acid of the sequence corresponding to ananchor position of a MHC binding motif to Nle or Nva; and synthesizing apeptide comprising the changed sequence. The synthesis can be forexample, chemical synthesis or any other synthetic method.

Some embodiments relate to T cell epitopes peptide analogue wherein theanalogue differs from a native epitope peptide by replacement of atleast one native residue corresponding to an anchor position of a MHCbinding motif with a Nle or Nva residue.

Further embodiments relate to methods to generate and resultingcompositions representing peptides that are immune active and carryunnatural amino acids at one or multiple MHC anchor residues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B summarize the substitutions that have been explored forSSX-2₄₁₋₄₉ analogs at each individual amino acid position for nonamersand decamers, respectively.

FIG. 2 is a schematic diagram of the methodology of a preferredembodiment for identifying analogs.

FIGS. 3A-B are tables showing the cross-reactivity and functionalavidity of SSX-2₄₁₋₄₉ analogs substituted at a single position.

FIGS. 4A-B are tables showing the cross-reactivity and functionalavidity of SSX-2₄₁₋₄₉ analogs substituted at two positions.

FIGS. 5A-D are tables showing the cross-reactivity and functionalavidity of SSX-2₄₁₋₄₉ analogs substituted at more than two positions.

FIG. 6 is a table showing the cross-reactivity and functional avidity ofSSX-2₄₁₋₄₉ decamer analogs encompassing the nominal 41-49 peptide.

FIG. 7 is a diagram showing the injection schedule of the SSX-2₄₁₋₄₉analogs.

FIG. 8 shows the activity of the SSX-2₄₁₋₄₉ A42V, A42L analogs andwild-type in lysis of tumor cells.

FIG. 9 shows the injections schedule for in vivo cytotoxicity studiesand ex vivo cytotoxicity studies as well as which SSX-2₄₁₋₄₉ analogpeptide was used for the boost.

FIG. 10 is a table showing the in vivo specific lysis results for anumber of the analogs as compared to a control (wild-type peptide) andEAA (Melan A 26-35).

FIG. 11 is a table showing the in vivo specific lysis results for anumber of the SSX-2₄₁₋₄₉ analogs as compared to a control (wild-typepeptide) and EAA as well as MHC binding and MHC stability.

FIG. 12 shows the percent specific lysis of tumor cells (624.38 humantumor cells) achieved following immunization with a number of analogs ascompared to a wild-type control.

Figure

FIGS. 13A-L summarize the substitutions that have been explored at eachindividual amino acid position for nonamers and decamers, respectively,as well as the results that were obtained.

FIG. 14 is a diagram showing the injection schedule used for analysisand testing of the NY-ESO-1 analogs.

FIGS. 15A-C show the specific elimination of target cells as measured byremoving the spleens and PBMC from challenged animals and measuring CFSEfluorescence by flow cytometry.

FIGS. 16A and B show the in vivo cytotoxicity against target cellscoated with wild-type peptide after boost with NY-ESO-1 analogs.

FIGS. 17A and B show an ex vivo analysis of the ability of the analogsto trigger enhanced immunity against the wild-type epitope as assessedby cytokine production.

FIG. 18 illustrates a protocol for validating the antigenicity of thePSMA₂₈₈₋₂₉₇ epitope, as well as the results of the testing.

FIGS. 19A-B are tables showing the cross-reactivity and functionalavidity of PSMA₂₈₈₋₂₉₇ analogs substituted at a single position.

FIGS. 20A-B are tables showing the cross-reactivity and functionalavidity of PSMA₂₈₈₋₂₉₇ analogs substituted at two positions.

FIGS. 21A-B are tables showing the cross-reactivity and functionalavidity of PSMA₂₈₈₋₂₉₇ analogs substituted at more than two positions.

FIG. 22 shows the immunogenicity of various PSMA₂₈₈₋₂₉₇ analogs measuredby Elispot.

FIG. 23 shows the results of an assay regarding the amplification ofanti-PSMA₂₈₈₋₂₉₇ response by the I297V analog measured by Elispot.

FIG. 24 shows the results of boosting with the I297V analog. The assayshowed that the boosting resulted in cytotoxic immunity against a PSMA⁺human tumor line.

FIG. 25 illustrates a protocol for validating the antigenicity of thePRAME₄₂₅₋₄₃₃ epitope, as well as the results of the testing.

FIGS. 26A-C are tables showing the cross-reactivity and functionalavidity of PRAME₄₂₅₋₄₃₃ analogs substituted at a single position.

FIGS. 27A-E are tables showing the cross-reactivity and functionalavidity of PRAME₄₂₅₋₄₃₃ analogs substituted at two positions.

FIG. 28 is a table showing the cross-reactivity and functional avidityof PRAME₄₂₅₋₄₃₃ analogs substituted at more than two positions.

FIG. 29 shows the immunogenicity of a PRAME₄₂₅₋₄₃₃ analog measured byElispot.

FIG. 30 shows the results of boosting with the L426Nva L433Nle analog.The assay showed that the boosting resulted in cytotoxic immunityagainst native epitope coated cells.

FIG. 31 shows a protocol for the in vivo evaluation of PRAME analogs, aswell as the results of the evaluation.

FIG. 32 shows a protocol for the ex vivo stimulation of cytokineproduction in analog induced, native epitope re-stimulated T cells.

FIG. 33 shows the results of boosting with the L426Nva L433Nle analog.The assay showed that the boosting resulted in cytotoxic immunityagainst a human tumor cell line.

FIG. 34 depicts a protocol for in vitro immunization to PRAME₄₂₅₋₄₃₃.

FIG. 35 shows the tetramer analysis results after in vitro immunizationwith PRAME₄₂₅₋₄₃₃ analogs.

FIG. 36 depicts the structure of the plasmid, pCTLR2, a plasmid thatexpresses the PRAME₄₂₅₋₄₃₃ epitope.

FIG. 37 shows the assay results for an experiment in which humor tumorcells were lysed by T cells primed with plasmid DNA and boosted withpeptides.

FIG. 38 shows the tetramer analysis results after plasmid prime withTyr₃₆₉₋₃₇₇ and peptide boost with the V377Nva analog.

FIG. 39 shows the percent specific lysis results of murine splenocytesachieved following immunization with Tyrosinase and Melan A analogs ascompared to controls.

FIG. 40 shows a schematic representation of an Tyrosinase analogimmunogenicity evaluation protocol.

FIG. 41 shows the immune response results against 624.38 cells contactedwith effector cells from HHD primed with plasmid and boosted withTyr₃₆₉₋₃₇₇ analogs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Peptides encompassing T cell epitopes are usually poor immunogens orimmune modulators due to one of multiple factors: a suboptimalpharmacokinetics profile, limited binding to MHC molecules (reducedK_(on) and increased K_(off)), decreased intrinsic recognition by Tcells present in the normal immune repertoire (e.g, through variousforms of tolerance). Various strategies have been pursued to improve theimmunologic properties of peptides, particularly the screening and useof peptides in which the sequence differs from the natural epitope. Suchanalogs are known by various names in the art, such as heteroclyticpeptides and altered peptide ligands (APL). The generation of suchanalogs has most often utilized amino acids from the standard set ofgenetically encoded residues (see for example Valmori, D. et al., J.Immunol. 160:1750-1758, 1998). Use of non-standard amino acids hastypically been associated with efforts to improve the biochemicalstability of the peptide (see, for example, Blanchet, J.-S. et al., J.Immunol. 167:5852-5861, 2001).

Generally, analogs can be categorized into the following two mainclasses: (1) modification of peptide anchor residues to achieve betterHLA binding profiles and higher immune responses, and (2) modificationof peptide anchor residues and TCR contact residues to circumvent T celltolerance for self-antigens.

Some embodiments relate to analogs that have at least one of thefollowing retained or improved properties, including but not limited to:

1. Cross-reactivity and functional avidity to TCR

2. Affinity for and stability of binding to MHC class I

3. In vivo effect on immunity assessed by cytotoxicity

4. In vivo effect on immunity assessed by ex vivo production ofIFN-gamma

5. Increased resistance to proteolysis.

Some embodiments relate to peptide sequences, including analogs, wherethe amino acids of the sequence are referred to with a positiondesignator, for example P1, P2, P3, PΩ, etc. In addition, the peptidesequences can be referred to as including a P0 and/or PΩ+1 designator.In some aspects, P0 can be X, XX, or XXX, where X is any amino acid orno amino acid. Similarly, in some aspects, PΩ+1 can be X, XX, or XXX,where X is any amino acid or no amino acid. Thus, for example, XXX canmean any combination of any amino acid residues or no amino acid. Thus,these embodiments can encompass polypeptides having up to threeadditional amino acids (with any combination of amino acid residues) onthe N-terminus or C-terminus of the specified sequence. Also, in someaspects, the embodiments can encompass no additional amino acids on theN-terminus or the C-terminus.

The MHC residues responsible for forming binding pockets, and whichbinding pockets accommodate epitope anchor residues and thus define thebinding specificity of the MHC molecule, are well understood in the art.One compilation of such information is found at the FIMM (FunctionalImmunology) web site at the hypertext transfer protocol (http://)“sdmc.lit.org.sg:8080/fimm/.” See also Schönbach C., Koh J. L. Y., ShengX., Wong L., and V. Brusic. FIMM, a database of functional molecularimmunology. Nucleic Acids Research, 2000, Vol. 28, No. 1 222-224; andSchönbach C., Koh J L, Flower D R, Wong L., and Brusic V. FIMM, adatabase of functional molecular immunology; update 2002. Nucleic AcidsResearch, 2002, Vol. 30, No. 1 226-229; each of which is herebyincorporated by reference in its entirety.

The phrase “liberation sequence” refers to a peptide comprising orencoding an epitope or an analog, which is embedded in a larger sequencethat provides a context allowing the epitope or analog to be liberatedby immunoproteasomal processing, directly or in combination withN-terminal trimming or other physiologic processes. In some aspects, theanalog or epitope can be designed or engineered.

Other embodiments relate to epitope arrays and other polypeptides thatinclude the epitope analog sequences that can be processed to liberatethe analog. Further embodiments relate to nucleic acids, particularlyDNA plasmids, encoding such polypeptides, or simply an analog, and theirexpression therefrom. The analogs, the polypeptides comprising them, andthe encoding nucleic acids can all be components of immunogeniccompositions, particularly compositions suitable for intralymphaticdelivery, all of which relate to further embodiments.

Peptide analogs with improved immunologic properties can be designed bymodifying the anchor residues involved in the interaction with MHCmolecules, so as to increase the binding and stabilize the formation ofMHC-peptide complexes. Such modifications can be guided by knowledge ofthe binding motif or preferred anchor residues of the restricting MHCmolecule. There further exist various rules, indexes and algorithms thatcan be used to predict the properties of analogs bearing varioussubstitutions with the limitation that the substitution is selected fromthe standard set of genetically encodable amino acids.

However, there are no databases or algorithms to predict the outcome ofreplacing anchor residues with non-standard amino acids and theirusefulness is previously not well explored. It is herein disclosed thatthe non-standard amino acids norleucine (Nle) and norvaline (Nva) can beadvantageously substituted into the anchor residue positions ofMHC-binding peptides. It is preferred that they be placed in a positionfavorably occupied by a hydrophobic or a large amino acid, especially I,L, or V.

MHC-binding motifs are generally defined in terms of preferred residueside chains at nominal positions within a span of 8 to 10 amino acids(see for example Rammensee et al., “MHC Ligands and Peptide Motifs,”(Molecular Biology Intelligence Unit), Springer-Verlag, Germany, 1997Landes Bioscience, Austin, Tex.; and Parker, et al., “Scheme for rankingpotential HLA-A2 binding peptides based on independent binding ofindividual peptide side-chains,” J. Immunol. 152:163-175. Websitealgorithms are also available which can be used to predict MHC binding.See for example, the world wide web page of Hans-Georg Rammensee, JuttaBachmann, Niels Emmerich, Stefan Stevanovic: SYFPEITHI: An InternetDatabase for MHC Ligands and Peptide Motifs (hypertext transfer protocolaccess via: syfpeithi.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm)and another is “bimas.dcrt.nih.gov/molbio/hla_bind.” For classI-restricted epitopes the C-terminal position, PΩ, is typically aprimary anchor. The 2^(nd) position, P2, is also often a primary anchoror, alternatively, P3 and/or P5 can serve this role. Positions P2through P7 have all been recognized as secondary or auxiliary anchorpositions for one or another MHC (see Rammensee et al., and see Table 6from U.S. Patent Application Publication No. 2003-0215425 (U.S. patentapplication Ser. No. 10/026,066, filed on Dec. 7, 2001, entitled EPITOPESYNCHRONIZATION IN ANTIGEN PRESENTING CELLS; which is incorporatedherein by reference in its entirety for all of its disclosure). Forclass II-restricted epitopes P1, P4, P6, P7, and P9 have been recognizedas anchor positions. The foregoing is intended as a general guide andshould be considered exemplary and not exhaustive or limiting. Manyanalyses and compilations of binding motifs, anchor residues, and thelike are available in the scientific and patent literature and over theinternet. Their conventions and results further provide those of skillin the art useful guide to the design of epitope analogs, when coupledwith the teaching herein.

The length of the peptide actually bound to the presenting MHC moleculecan be longer than the nominal motif sequence. The ends of the bindingcleft for class II MHC molecules are open so that the bound peptide canbe extended at either end of the core motif. In contrast the bindingcleft is closed at both ends in class I MHC molecules so that the endsof the bound peptide must generally correspond to the motif, howeversignificant variation in length can be accommodated through bulging andfolding of the central region of the bound peptide, so that peptides ofup to at least about 14 amino acids in length can be presented (see forexample Probst-Kepper, M. et al., J. Immunol. 173:5610-5616, 2004).

Epitope analogs can have improved K_(on) and K_(off) related to theinteraction with class I MHC molecules, as well as preserved orincreased interaction with T cell receptors recognizing the originalepitope, modified or improved in vivo or ex vivo activity reflected inenhanced expansion of specific T cell populations, improved cytokineproduction by specific T cells, or in vivo or in vitro cytotoxicityagainst targets carrying natural epitopes, mediated by T cells thatreacted with the peptide. In addition, such analogs may interact in amore optimal fashion with multiple distinct MHC class 1 molecules.

Such peptide analogs with improved immune properties may encompass oneor multiple substitutions, including one or multiple non-standard aminoacids. Among non-standard amino acids, substitutions for primary anchorresidues consisting of norvaline or norleucine are preferred since, asexemplified below, they can not only improve on the interaction with MHCclass I, but can also preserve cross-reactivity with TCR specific forthe native epitope and show improved in vivo immune profile. Morespecifically, mutating the P2 amino acid residue from A, L or V tonorvaline or norleucine improved immune properties and is thuspreferred. In addition, modifying the C terminal residue to norvaline orpreferably norleucine, improved immune features of the analogs. Inaddition, analogs that encompass multiple substitutions at primaryand/or secondary anchor residues including norvaline and/or norleucineat P2 or PΩ, can be associated with improved immune properties.

Certain uses of norvaline (Nva) and norleucine (Nle) are mentioned inU.S. Pat. No. 6,685,947, PCT Publication Nos. WO 03/076585 A2 and WO01/62776 A1 and U.S. Patent Publication No. 20040253218A1. None of thesereferences teaches the general usefulness of Nva or Nle substituted atan anchor position of a MHC-biding peptide to improve an immunologicalproperty. The '218 publication teaches that the substituted residuesshould be incorporated at TCR-interacting positions and not atMHC-interacting positions:

-   -   In still another embodiment of the invention, the peptide is an        analog of a peptide derived from an NS-specific antigen that is        immunogenic but not encephalitogenic. The most suitable peptides        for this purpose are those in which an encephalitogenic        self-peptide is modified at the T-cell receptor (TCR) binding        site and not at the MHC binding site(s), so that the immune        response is activated but not anergized (Karin et al, 1998;        Vergelli et al, 1996).

HLA-A2.1-restricted peptides incorporating Nle disclosed in the '776publication are derived from CEA, p53, and MAGE-3. In the CEA peptideI(Nle)GVLVGV (SEQ ID NO. 143) and the p53 peptide S(Nle)PPPGTRV (SEQ IDNO. 144), Nle is present at the P2 position. No teaching about thegeneral usefulness of norleucine is provided and no disclosure isprovided indicating how or if these substitutions altered the propertiesof the analogs as compared to the native sequence.

Some of the instant embodiments relate to epitope analogs thatincorporate Nva and/or Nle at a position promoting binding to MHC. Someembodiments specifically exclude the use Nle and/or Nva inHLA-A2.1-restricted epitopes, HLA-A2.1 epitopes from CEA, p53, and/orMAGE-3, or other peptides derived from MAGE-3, CEA, and/or p53. In someembodiments, one or more of the specific sequences as disclosed in theabove referenced patent references are specifically excluded. Otherexemplary embodiments include the use of Nle and/or Nva at P3, P5,and/or PΩ anchor positions, at an auxiliary anchor position, to make ananalog of a non-A2- or non-A2.1-HLA restricted epitope, in an anchorposition of a peptide that is not derived from an oncogene or oncofetalprotein, and in an anchor position of a peptide derived from a CTantigen.

In general, such analogs may be useful for immunotherapy and/orprophylaxis of various diseases such as infectious, cancerous orinflammatory, as single agents or in combination therapies, due to theiroptimized interaction with MHC molecules and T cell receptors, key toonset and regulation of immune responses.

Analog Production

The analogs may be produced using any method known to one of skill inthe art, including manufacturing the peptides using a method of peptidesynthesis or expressing nucleic acids that code for the desired peptideanalogs. Thus, when the analogs include one or more non-standard aminoacids, it is more likely that they will be produced by a method ofpeptide production. When the analogs include only one or moresubstitutions with standard amino acids, they may be expressed from anexpression vector using any method known to one of skill in the art.Alternatively, the peptides may be expressed using a method of genetherapy.

Analog Testing

The usefulness and/or the activity of the analogs was identified. Inthis way useful and/or improved analogs can be identified. To be useful,an analog may not necessarily be found to be improved in the identifiedassays. Thus, a useful peptide may contain other properties such asbeing useful in a tolerized patient or resistant to proteolysis. To beimproved, a peptide can be found to have a clear improvement in bindingto the TCR, binding to the MHC molecule, and an improved immune responseor any other biological activity. To be useful, the peptide may be foundnot to be improved when using a murine test system, but because of thedifferences in the human immune system, may be improved when tested in ahuman. Alternatively, the usefulness may stem from a potential to breaktolerance in a tolerized human. Alternatively, the usefulness may stemfrom the ability to use the peptide as a base for further substitutionsto identify improved analogs.

In order to evaluate usefulness, improved properties and to compare theanalogs in any way to the wild-type, one or more of the following assayswere conducted: peptide binding affinity for HLA-A*0201;peptide-HLA-A*0201 complex stability assay; cross-reactivity assay(recognition of peptide analogs by wild-type peptide specific CTL orrecognition of wild-type peptide by CTL generated using peptideanalogs); immunogenicity assays, such as an IFN-γ secretion assay, acytotoxicity assay, and/or an Elispot assay; antigenicity assays such asan in vitro tumor cell lysis assay, an ex vivo tumor cell lysis, and anin vivo tumor cell lysis; and proteolysis assays to identify increasedresistance to proteolysis. Details of exemplary assays are presented inthe Examples.

Uses of the Analogs

Useful methods for using the disclosed analogs in inducing, entraining,maintaining, modulating and amplifying class I MHC-restricted T cellresponses, and particularly effector and memory CTL responses toantigen, are described in U.S. patent application Ser. Nos. 09/380,534and 09/776,232 both entitled A METHOD OF INDUCING A CTL RESPONSE; U.S.Provisional Application No. 60/479,393, filed on Jun. 17, 2003, entitledMETHODS TO CONTROL MHC CLASS I-RESTRICTED IMMUNE RESPONSE; and U.S.patent application Ser. No. 10/871,707 (Pub. No. 2005 0079152) andProvisional U.S. Patent Application No. 60/640,402 filed on Dec. 29,2004, both entitled METHODS TO ELICIT, ENHANCE AND SUSTAIN IMMUNERESPONSES AGAINST MHC CLASS I-RESTRICTED EPITOPES, FOR PROPHYLACTIC ORTHERAPEUTIC PURPOSE. The analogs can also be used in research to obtainfurther optimized analogs. Numerous housekeeping epitopes are providedin U.S. application Ser. Nos. 10/117,937, filed on Apr. 4, 2002 (Pub.No. 20030220239 A1), and 10/657,022 (2004-0180354), and in PCTApplication No. PCT/US2003/027706 (Pub. No. WO04022709A2), filed on Sep.5, 2003; and U.S. Provisional Application Nos. 60/282,211, filed on Apr.6, 2001; 60/337,017, filed on Nov. 7, 2001; 60/363,210 filed on Mar. 7,2002; and 60/409,123, filed on Sep. 5, 2002; each of which applicationsis entitled EPITOPE SEQUENCES. The analogs can further be used in any ofthe various modes described in those applications. Epitope clusters,which may comprise or include the instant analogs, are disclosed andmore fully defined in U.S. patent application Ser. No. 09/561,571, filedon Apr. 28, 2000, entitled EPITOPE CLUSTERS. Methodology for using anddelivering the instant analogs is described in U.S. patent applicationSer. Nos. 09/380,534 and 09/776,232 (Pub. No. 20020007173 A1), and inPCT Application No. PCTUS98/14289 (Pub. No. WO9902183A2) each entitled AMETHOD OF INDUCING A CTL RESPONSE. Beneficial epitope selectionprinciples for such immunotherapeutics are disclosed in U.S. patentapplication Ser. No. 09/560,465, filed on Apr. 28, 2000, 10/026,066(Pub. No. 20030215425 A1), filed on Dec. 7, 2001, and 10/005,905 filedon Nov. 7, 2001, all entitled EPITOPE SYNCHRONIZATION IN ANTIGENPRESENTING CELLS; 09/561,074 entitled METHOD OF EPITOPE DISCOVERY;09/561,571, filed Apr. 28, 2000, entitled EPITOPE CLUSTERS; 10/094,699(Pub. No. 20030046714 A1), filed Mar. 7, 2002, entitledANTI-NEOVASCULATURE PREPARATIONS FOR CANCER; application Ser. Nos.10/117,937 (Pub. No. 20030220239 A1) and PCTUS02/11101 (Pub. No.WO02081646A2), both filed on Apr. 4, 2002, and both entitled EPITOPESEQUENCES; and application Ser. Nos. 10/657,022 and PCT Application No.PCT/US2003/027706 (Pub. No. WO04022709A2), both filed on Sep. 5, 2003,and both entitled EPITOPE SEQUENCES. Aspects of the overall design ofvaccine plasmids are disclosed in U.S. patent application Ser. No.09/561,572, filed on Apr. 28, 2000, entitled EXPRESSION VECTORS ENCODINGEPITOPES OF TARGET-ASSOCIATED ANTIGENS and 10/292,413 (Pub. No.20030228634 A1), filed on Nov. 7, 2002, entitled EXPRESSION VECTORSENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIRDESIGN; 10/225,568 (Pub No. 2003-0138808) filed on Aug. 20, 2002, PCTApplication No. PCT/US2003/026231 (Pub. No. WO 2004/018666) filed onAug. 19, 2003, both entitled EXPRESSION VECTORS ENCODING EPITOPES OFTARGET-ASSOCIATED ANTIGENS; and U.S. Pat. No. 6,709,844, entitledAVOIDANCE OF UNDESIRABLE REPLICATION INTERMEDIATES IN PLASMIDPROPAGATION. Specific antigenic combinations of particular benefit indirecting an immune response against particular cancers are disclosed inProvisional U.S. patent Application No. 60/479,554 filed on Jun. 17,2003 and U.S. patent application Ser. No. 10/871,708 filed on Jun. 17,2004 and PCT Patent Application No. PCT/US2004/019571 (Pub. No. WO2004/112825), all entitled COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS INVACCINES FOR VARIOUS TYPES OF CANCERS. Antigens associated with tumorneovasculature (e.g., PSMA, VEGFR2, Tie-2) are also useful in connectionwith cancerous diseases, as is disclosed in U.S. patent application Ser.No. 10/094,699 (Pub. No. 20030046714 A1), filed Mar. 7, 2002, entitledANTI-NEOVASCULATURE PREPARATIONS FOR CANCER. Methods to trigger maintainand manipulate immune responses by targeted administration of biologicalresponse modifiers are disclosed U.S. Provisional Application No.60/640,727, filed on Dec. 29, 2004. Methods to bypass CD4+ cells in theinduction of an immune response are disclosed in U.S. ProvisionalApplication No. 60/640,821, filed on Dec. 29, 2004. Exemplary diseases,organisms and antigens and epitopes associated with target organisms,cells and diseases are described in U.S. application Ser. No. 09/776,232(Pub. No. 20020007173), filed Feb. 2, 2001 and entitled METHOD OFINDUCING A CTL RESPONSE. Exemplary methodology is found in U.S.Provisional Application No. 60/580,969, filed on Jun. 17, 2004, and U.S.patent application Ser. No. 11/155,288, filed on Jun. 17, 2005, bothentitled COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN DIAGNOTISTICS FORVARIOUS TYPES OF CANCERS. Methodology and compositions are alsodisclosed in U.S. Provisional Application No. 60/640,598, filed on Dec.29, 2004, entitled COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS INCOMPOSITIONS FOR VARIOUS TYPES OF CANCER. The integration of diagnostictechniques to assess and monitor immune responsiveness with methods ofimmunization including utilizing the instant analogs is discussed morefully in Provisional U.S. Patent Application No. 60/580,964 filed onJun. 17, 2004 and U.S. patent application Ser. No. 11/155,928, filed onJun. 17, 2005, both entitled IMPROVED EFFICACY OF ACTIVE IMMUNOTHERAPYBY INTEGRATING DIAGNOSTIC WITH THERAPEUTIC METHODS. The immunogenicpolypeptide encoding vectors are disclosed in U.S. patent applicationSer. No. 10/292,413 (Pub. No. 20030228634 A1) filed on Nov. 7, 2002entitled EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATEDANTIGENS AND METHODS FOR THEIR DESIGN, and in U.S. ProvisionalApplication No. 60/691,579, filed on Jun. 17, 2005, entitled METHODS ANDCOMPOSITIONS TO ELICIT MULTIVALENT IMMUNE RESPONSES AGAINST DOMINANT ANDSUBDOMINANT EPITOPES, EXPRESSED ON CANCER CELLS AND TUMOR STROMA.Additional useful disclosure, includes methods and compositions ofmatter is found in U.S. Provisional Application No. 60/691,581, filed onJun. 17, 2005, entitled MULTIVALENT ENTRAIN-AND-AMPLIFYIMMUNOTHERAPEUTICS FOR CARCINOMA. Further methodology, compositions,peptides, and peptide analogs are disclosed in U.S. ProvisionalApplication Nos. 60/581,001 and 60/580,962, both filed on Jun. 17, 2004,and respectively entitled “SSX-2 PEPTIDE ANALOGS” and “NY-ESO PEPTIDEANALOGS.” All of these applications mentioned in this paragraph arehereby incorporated by reference in their entirety for all that theyteach. Additional analogs, peptides and methods are disclosed in U.S.patent application Ser. No. 11/155,929, entitled SSX-2 PEPTIDE ANALOGS;and U.S. Prov. Application No. 60/691,889, entitled EPITOPE ANALOGS; andPCT Application No. 2007-516810, entitled EPITOPE ANALOGS; all filed onJun. 17, 2005. As an example, without being limited thereto thereferences are incorporated by reference for what they teach about classI MHC-restricted epitopes, analogs, the design of analogs, uses ofepitopes and analogs, methods of using and making epitopes, and thedesign and use of nucleic acid vectors for their expression.

Antigens

There are many antigens, epitopes of which can be recognized by T cellsin an MHC-restricted manner, for which manipulation of an immuneresponse directed against them has therapeutic or prophylacticpotential. The principles for making analogs of MHC-binding peptidesdescribed herein are generally applicable to any of these antigens andtheir epitopes. A particular focus of the present disclosure is epitopesfrom the tumor-associated antigens (TuAA) SSX-2, NY-ESO-1, PRAME, PSMA,tyrosinase, and Melan-A.

SSX-2, also know as Hom-Mel-40, is a member of a family of highlyconserved cancer-testis antigens (Gure, A. O. et al. Int. J. Cancer72:965-971, 1997, which is hereby incorporated by reference in itsentirety). Its identification as the TuAA antigen is taught in U.S. Pat.No. 6,025,191 entitled ISOLATED NUCLEIC ACID MOLECULES THAT ENCODE AMELANOMA SPECIFIC ANTIGEN AND USES THEREOF, which is hereby incorporatedby reference in its entirety. Cancer-testis antigens are found in avariety of tumors, but are generally absent from normal adult tissuesexcept testis. SSX-2 is expressed in many different types of tumors,including synovial sarcomas, melanoma, head and neck cancers, breast,colon and ovarian cancers. In addition to its widespread expression in avariety of cancers, it is also immunogenic in patients with late stagedisease. Further, there is evidence of spontaneous humoral and cellularimmune responses towards this antigen in metastatic tumor patients(Ayyoub M, et al., Cancer Res. 63(17): 5601-6, 2003; Ayyoub M, et al. J.Immunol. 168(4): 1717-22, 2002), which is incorporated herein byreference in its entirety. Two HLA-A2 restricted T cell epitopes havebeen identified recently using reverse T-cell immunology, namelySSX-2₄₁₋₄₉ (Ayyoub M, et al. J. Immunol. 168(4): 1717-22, 2002; U.S.Pat. No. 6,548,064, entitled ISOLATED PEPTIDES CONSISTING OF AMINO ACIDSEQUENCES FOUND 1N SSX OR NY-ESO-1 MOLECULES, THAT BIND TO HLA MOLECULE;U.S. patent application Ser. No. 10/117,937, entitled EPITOPE SEQUENCES)and SSX-2₁₀₃₋₁₁₁ (Wagner C, et al. Cancer Immunity 3:18, 2003), each ofwhich is incorporated herein by reference in its entirety. The C-terminiof both epitopes can be efficiently generated by in vitro proteasomedigestion. Isolated HLA-A*0201/SSX-2₄₁₋₄₉ multimer⁺ CD8⁺ T cells fromtumor-infiltrated lymph nodes of SSX-2 positive patients exhibited highfunctional avidity and can effectively recognize SSX-2 positive tumors;however, the spontaneously occurring immunological responses were notsufficient for stopping tumor growth, possibly because these immuneresponse did not develop until fairly late in the disease progression,and the activated T cells were not numerous enough. U.S. Pat. No.6,548,064 (which is incorporated herein by reference in its entirety)further describes substituting a T or A residue at both the P2 and PΩposition of an SSX-2 epitope.

NY-ESO-1 is a cancer-testis antigen found in a wide variety of tumorsand is also known as CTAG-1 (Cancer-Testis Antigen-1) and CAG-3 (CancerAntigen-3). NY-ESO-1 as a tumor-associated antigen (TuAA) is disclosedin U.S. Pat. No. 5,804,381 entitled ISOLATED NUCLEIC ACID MOLECULEENCODING AN ESOPHAGEAL CANCER ASSOCIATED ANTIGEN, THE ANTIGEN ITSELF,AND USES THEREOF that is hereby incorporated by reference in itsentirety. A paralogous locus encoding antigens with extensive sequenceidentity, LAGE-1a/s and LAGE-1b/L, have been disclosed in publiclyavailable assemblies of the human genome, and have been concluded toarise through alternate splicing. Additionally, CT-2 (or CTAG-2,Cancer-Testis Antigen-2) appears to be either an allele, a mutant, or asequencing discrepancy of LAGE-1b/L. Due to the extensive sequenceidentity, many epitopes from NY-ESO-1 can also induce immunity to tumorsexpressing these other antigens. The proteins are virtually identicalthrough amino acid 70. From 71-134 the longest run of identities betweenNY-ESO-1 and LAGE is 6 residues, but potentially cross-reactivesequences are present. And from 135-180 NY-ESO and LAGE-1a/s areidentical except for a single residue, but LAGE-1b/L is unrelated due tothe alternate splice. The CAMEL and LAGE-2 antigens appear to derivefrom the LAGE-1 mRNA, but from alternate reading frames, thus givingrise to unrelated protein sequences. More recently, GenBank AccessionAF277315.5 (which is incorporated herein by reference in its entirety),Homo sapiens chromosome X clone RP5-865E18, RP5-1087L19, completesequence, reports three independent loci in this region that are labeledas LAGE1 (corresponding to CTAG-2 in the genome assemblies), plusLAGE2-A and LAGE2-B (both corresponding to CTAG-1 in the genomeassemblies).

NY-ESO-1₁₅₇₋₁₆₅ is identified as an HLA-A2 restricted epitope in U.S.Pat. No. 6,274,145 entitled ISOLATED NUCLEIC ACID MOLECULE ENCODINGCANCER ASSOCIATED ANTIGEN, THE ANTIGEN ITSELF, AND USES THEREOF, andU.S. patent application Ser. No. 10/117,937 (Pub. No. 20030220239)entitled EPITOPE SEQUENCES reports that this C-terminus is generated bythe housekeeping proteasome in an in vitro assay. Analogs substitutingA, V, L, I, P, F, M, W, or G at PΩ, alone or in combination with A atanother position, are disclosed in U.S. Pat. Nos. 6,417,165 and6,605,711, both entitled NY-ESO-1-PEPTIDE DERIVATIVES, AND USES THEREOF.The references described in this paragraph are incorporated herein byreference in their entirety.

PRAME, also know as MAPE, DAGE, and OIP4, was originally observed as amelanoma antigen. Subsequently, it has been recognized as a CT antigen,but unlike many CT antigens (e.g., MAGE, GAGE, and BAGE) it is expressedin acute myeloid leukemias. PRAME is a member of the MAPE family whichconsists largely of hypothetical proteins with which it shares limitedsequence similarity. The usefulness of PRAME as a TuAA is taught in U.S.Pat. No. 5,830,753 entitled ISOLATED NUCLEIC ACID MOLECULES CODING FORTUMOR REJECTION ANTIGEN PRECURSOR DAGE AND USES THEREOF, which is herebyincorporated by reference in its entirety. U.S. patent application Ser.No. 10/181,499, entitled METHODS FOR SELECTING AND PRODUCING T CELLPEPTIDE EPITOPES AND VACCINES INCORPORATING SAID SELECTED EPITOPES(which is incorporated herein by reference in its entirety) identifies avariety of potential epitopes, including PRAME₄₂₅₋₄₃₃, using in vitrodigestion with immunoproteasome.

PSMA (prostate-specific membranes antigen), a TuAA described in U.S.Pat. No. 5,538,866 entitled “PROSTATE-SPECIFIC MEMBRANES ANTIGEN” whichis hereby incorporated by reference in its entirety, is expressed bynormal prostate epithelium and, at a higher level, in prostatic cancer.It has also been found in the neovasculature of non-prostatic tumors.PSMA can thus form the basis for vaccines directed to both prostatecancer and to the neovasculature of other tumors. This later concept ismore fully described in U.S. Patent Publication No. 20030046714; PCTPublication No. WO 02/069907; and a provisional U.S. Patent applicationNo. 60/274,063 entitled ANTI-NEOVASCULAR VACCINES FOR CANCER, filed Mar.7, 2001, and U.S. application Ser. No. 10/094,699, filed on Mar. 7,2002, entitled “ANTI-NEOVASCULAR PREPARATIONS FOR CANCER,” each of whichare hereby incorporated by reference in its entirety. The teachings andembodiments disclosed in said publications and applications arecontemplated as supporting principals and embodiments related to anduseful in connection with the present invention. Briefly, as tumors growthey recruit ingrowth of new blood vessels. This is understood to benecessary to sustain growth as the centers of unvascularized tumors aregenerally necrotic and angiogenesis inhibitors have been reported tocause tumor regression. Such new blood vessels, or neovasculature,express antigens not found in established vessels, and thus can bespecifically targeted. By inducing CTL against neovascular antigens thevessels can be disrupted, interrupting the flow of nutrients to (andremoval of wastes from) tumors, leading to regression.

Alternate splicing of the PSMA mRNA also leads to a protein with anapparent start at Met₅₈, thereby deleting the putative membrane anchorregion of PSMA as described in U.S. Pat. No. 5,935,818 entitled“ISOLATED NUCLEIC ACID MOLECULE ENCODING ALTERNATIVELY SPLICEDPROSTATE-SPECIFIC MEMBRANES ANTIGEN AND USES THEREOF” which is herebyincorporated by reference in its entirety. A protein termed PSMA-likeprotein, Genbank accession number AF261715, which is hereby incorporatedby reference in its entirety, is nearly identical to amino acids 309-750of PSMA and has a different expression profile. Thus the more preferredepitopes are those with an N-terminus located from amino acid 58 to 308.PSMA₂₈₈₋₂₉₇ was identified as possessing an HLA-A2 binding motif in WO01/62776, entitled HLA BINDING PEPTIDES AND THEIR USES, which is herebyincorporated by reference in its entirety. Its production in vitro bydigestion with housekeeping proteasome and actual binding to HLA-A2 wasdisclosed in U.S. Patent Application Publication No. 2003-0220239entitled EPITOPE SEQUENCES.

Tyrosinase is a melanin biosynthetic enzyme that is considered one ofthe most specific markers of melanocytic differentiation. Tyrosinase isexpressed in few cell types, primarily in melanocytes, and high levelsare often found in melanomas. The usefulness of tyrosinase as a TuAA istaught in U.S. Pat. No. 5,747,271 entitled “METHOD FOR IDENTIFYINGINDIVIDUALS SUFFERING FROM A CELLULAR ABNORMALITY SOME OF WHOSE ABNORMALCELLS PRESENT COMPLEXES OF HLA-A2/TYROSINASE DERIVED PEPTIDES, ANDMETHODS FOR TREATING SAID INDIVIDUALS” which is hereby incorporated byreference in its entirety.

Melan-A, also called MART-1 (Melanoma Antigen Recognized by T cells), isanother melanin biosynthetic protein expressed at high levels inmelanomas. The usefulness of Melan-A/MART-1 as a TuAA is taught in U.S.Pat. Nos. 5,874,560 and 5,994,523 both entitled MELANOMA ANTIGENS ANDTHEIR USE IN DIAGNOSTIC AND THERAPEUTIC METHODS, as well as U.S. Pat.No. 5,620,886, entitled ISOLATED NUCLEIC ACID SEQUENCE CODING FOR ATUMOR REJECTION ANTIGEN PRECURSOR PROCESSED TO AT LEAST ONE TUMORREJECTION ANTIGEN PRESENTED BY HLA-A2, all of which are herebyincorporated by reference in their entirety. The immunodominant HLA-A2restricted epitope from this TuAA is Melan-A₂₆₋₃₅. It has been shown tobe produced by the housekeeping proteasome (Morel, S. et al. Immunity12:107-117, 2000), which is hereby incorporated by reference in itsentirety. Various analogs incorporating standard amino acids, includingan improved analog substituting L at P2, are disclosed in U.S. Pat. No.6,025,470, entitled ISOLATED NONA- AND DECAPEPTIDES WHICH BIND TO HLAMOLECULES, AND THE USE THEREOF, which is hereby incorporated byreference in its entirety. The use of analogs incorporating non-standardamino acids with a primary goal of improving biochemical stability isreported by Blanchet, J.-S. et al., J. Immunol. 167:5852-5861, 2001,which is hereby incorporated by reference in its entirety.

SSX-2 41-49 Analogs

As noted above, the natural immune response to SSX-2 in cancer patients,including the response to SSX-2₄₁₋₄₉, may not be effective incontrolling cancer. Additionally, wild-type SSX-2₄₁₋₄₉ is only amoderately immunogenic peptide that can further limit its clinicalpotential. Stronger SSX-2 specific immune responses induced by the useof superagonist analogs results in clinical benefits for patients withSSX-2 positive tumor.

Thus, in one embodiment, the analogs can be used in compositions tostimulate the immune response of a subject to mount an immune responseagainst a target cell displaying the target antigen. The embodiment iscontemplated to have utility in the treatment and prevention ofneoplastic and viral disease.

Since the wild-type SSX-2₄₁₋₄₉ is only a moderately immunogenic peptidethat may prevent it from eliminating tumors effectively in vivo, amethod was used to de novo design SSX-2₄₁₋₄₉ variants that were morepotent or had a variety of improved properties. By using a moreimmunogenic SSX-2 analog peptide, it was possible to stimulate astronger immune response and/or to amplify the naturally occurringimmune response to achieve a better chance of clinical response. Thus,the binding properties (affinity and HLA-A*0201/peptide complexesstability), immunogenicity, antigenicity and cross-reactivity to thewild-type epitope were analyzed for each of the analogs to identify animproved property. In some embodiments, by improved property it is meantgenerally, that the analog can be better used for some purpose than thewild-type. Thus, the analog need not exhibit improved binding,stability, or activity to be improved and may even show a reducedability to mediate certain parts of the process, but still be improvedfor use in another way. For example, analogs that retain some activity,but not all activity may be better in human systems that are tolerizedto the wild-type antigen.

Previously, modifications of natural tumor-associated peptide epitopesby incorporating favorable anchor residues have generated analogs withimproved binding profiles with HLA molecules and enhancedimmunogenicity. One of the most successful examples is the A27L peptideanalog of Melan-A 26-35 epitope. Valmori et al., “Enhanced generation ofspecific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogs,” J Immunol. 1998, 160(4): 1750-8; which ishereby incorporated by reference in its entirety. The original epitopefailed to form a stable complex with HLA-A2 molecules since it lacksoptimum anchor amino acid residue at position 2. The modified A27L MelanA 26-35 peptide analog has demonstrated unequivocally increased bindingprofiles with HLA-A2 molecules and greater immunogenicity than itswild-type counterpart. Immunizing patients with this analog can generatestrong T cell immune responses that were able to recognize the wild-typeepitope presented at the cell surfaces. Similar modifications have beenobtained successfully with many other tumor-associated epitopes such asGP100 209-217 (Parkhurst et al., “Improved induction ofmelanoma-reactive CTL with peptides from the melanoma antigen gp100modified at HLA-A*0201-binding residues,” J Immunol. 1996, 157(6):2539-48; which is hereby incorporated by reference in its entirety),Her-2369-377 (Vertuani et al., “Improved immunogenicity of animmunodominant epitope of the HER-2/neu protooncogene by alterations ofMHC contact residues,” J. Immunol. 2004, 172(6): 3501-8; which is herebyincorporated by reference in its entirety).

Up to this point no SSX-2₄₁₋₄₉ analogs have been designed and studiedeven though they hold great promise for development of SSX-2-basedvaccines to treat a variety of cancers, particularly SSX-2 positivecancers and/or tumors. Thus, methods are disclosed herein that can beused for the identification and production of analogs to a Synovialsarcoma X breakpoint 2 (SSX-2) wild-type sequence. Using the methods, apanel of 95 novel SSX-241-49 analogs based on the wild-type sequencefrom amino acids 241-249 were identified with a variety of improvedproperties. The improved properties include, but are not limited to,binding to class I MHC and T cell receptor (TCR) molecules, andbiological responses such as IFN-γ secretion, cytotoxicity, and tumorcell lysis. Peptides with improved potency that retainedcross-reactivity with the wild-type epitope were identified. Among theseanalogs, some have been demonstrated to be the superagonist variants ofthe wild-type SSX-241-49 peptide, some of which analogs have been shownto have much higher affinity with HLA-A*0201 molecule, and thepeptide-HLA complex possessed extended stability. When the mice wereimmunized with these analogs, they were able to induce enhanced CTLimmune responses in HHD transgenic mice. The resulting CTLs couldeffectively lyse A2+ and SSX-2+ tumor cell lines both in vivo and invitro, which indicated that the CTLs generated using the analogs wereable to recognize the wild-type SSX-241-49 epitope that naturallypresented at the cell surfaces. In comparison with the wild-typeSSX-241-49 epitope, the analogs are better candidates for thedevelopment of cancer vaccines.

Embodiments include families of one or more peptides of 9 or 10 aminoacids in length related by sequence to amino acids 41-49 of the humancancer testis (CT) antigen SSX-2 (SSX-2₄₁₋₄₉). The individual peptideembodiments have one to several defined amino acid substitutions in thewild-type sequence. The substituted amino acids are, variously, othermembers of the standard set of amino acids commonly genetically encoded,derivatives thereof, their D-stereoisomers, or other non-standardL-amino acids. These analogs are useful for investigating theinteraction of the wild-type epitope with class I MHC and TCR moleculesand other components of the immune response, and for designingadditional analogs with further optimized immunologic properties. Someembodiments of the analogs have at least similar immunologic propertiesto the wild-type epitope in the HLA-transgenic mouse model in which theyhave been tested. Such peptides can be useful in humans, as SSX-2 is aself-antigen to which a degree of tolerance may be expected, and theamino acid differences of the analogs can help to stimulate populationsof T cells that have avoided negative selection but are cross-reactivewith the wild-type epitope. Various peptide embodiments can have one ormore improved immunologic properties in that they possess greateraffinity for MHC or greater stability of binding to MHC, elicit greatercytokine production or require lower peptide concentrations to elicitsimilar cytokine production from T cells that recognize the wild-typeepitope, are more immunogenic, can induce or amplify a cross-reactivecytolytic response to the wild-type epitope, or can break tolerance.

In one embodiment, the analogs can have at least one substitution at aresidue selected from the group consisting of, P1, P2, P4, P6, P8, P9and P10. In a further embodiment, the analogs can have at least twosubstitutions at residues selected from the group consisting of: P1, P2,P4, P6, P8, P9 and P10. In a further embodiment, the analogs can have atleast three substitutions at residues selected from the group consistingof: P1, P2, P4, P6, P8, P9 and P10. In a further embodiment, the analogscan have substitutions at positions P2 and P9. In a further embodiment,the peptides can have substitutions at residues P1, P2, and P9. In afurther embodiment, the peptide analogs can have substitutions atresidues P1, P2, and P4. In a further embodiment, the peptide analogscan have substitutions at residues P1, P2, and P6. In a furtherembodiment, the peptide analogs can have substitutions at residues P1,P2, and P8. In one embodiment, two substitutions can produce improvedproperties. In a further embodiment, one substitution can produceimproved properties. In a further embodiment, three substitutions canproduce improved properties. In a further embodiment, the one or moresubstitutions can produce improved properties but are still recognizedby a TCR that recognizes the wild-type sequence (still cross-react withthe wild-type sequence).

One embodiment relates to epitope arrays and other polypeptidescomprising the epitope analog sequences that can be processed toliberate the analog. Further embodiments relate to nucleic acids,particularly DNA plasmids, encoding such polypeptides, or simply ananalog, and their expression therefrom. The analogs, the polypeptidescomprising them, and the encoding nucleic acids can all be components ofimmunogenic compositions, particularly compositions suitable forintralymphatic delivery, that constitute further embodiments.

Analog Design

Embodiments relate to the SSX-2₄₁₋₄₉ peptide which contain substitutionsof the sequence KASEKIFYV (SEQ ID NO. 1) (See FIG. 1). In a furtherembodiment, the analog can be generally an analog of the SSX-2₄₁₋₅₀decamer peptide with the sequence KASEKIFYVY (SEQ ID NO. 79). Theresidues or amino acids that make up the peptide are referred to hereinas P1-P9 or P1-P10 to designate the position within the peptide asnumbered from the N- to the C-terminus, P1 corresponding to theN-terminal Lysine and P9 corresponding to the C-terminal Valine in thenonamer. Alternatively, the residues may be referred to by the primaryactivity of the molecule that they are involved in. For example, residueP2 is described as the N-terminal primary anchor molecule, while P9 (orP10 in the decamer) is described as the primary C-terminal anchor.Residues P4, P6 and P8 are primarily involved in TCR interactions.Substitutions can use any amino acids, including standard andnon-standard amino acids, known to one of skill in the art. A number ofexemplary amino acids are disclosed herein, however, the substitutionsdisclosed herein are not meant to be a list that includes all imaginedsubstitutions, but are exemplary of the substitutions that are possible.One of skill in the art may find a number of other non-standard aminoacids in catalogs and references that may be purchased or chemicallyproduced for use with the analogs herein.

A number of possible analogs were produced by modification of peptideanchor residues to achieve better HLA binding profiles and higher immuneresponses, including at the N-terminal primary anchor (P2 position), atthe N-terminal secondary anchor (P1 position), at the N-terminal primaryand secondary anchor (P1 and P2 positions), and at the N-terminalprimary/secondary anchor (P1 and P2 positions) and C-terminal primaryanchor (P9 position). Further, peptides with modifications at the anchorresidues and TCR contact residues were produced to circumvent T celltolerance for self-antigens, these modifications included modificationsat the N-terminal primary/secondary anchor (P1 and P2 positions) andsecondary TCR recognition sites (P4, P6 and/or P8 positions),modifications at the N-terminal primary/secondary anchors (P1 and P2position), and modifications at the C-terminal primary anchor (P9) andat secondary TCR recognition sites (P4, P6 and/or P8 positions).Further, decamer analogs were produced.

The choice of which residues would best produce analogs with improvedproperties involved analysis of studies of MHC peptide interactions,studies of TCR peptide interactions and previous analogs that were knownin the art. Some residues are primarily involved in a specificinteraction and some are secondarily or even tertiarily involved. Thus,the knowledge of how the residues are involved in the binding to thesemolecules was involved in the analysis. Further some of the wild-typeresidues are preferred, meaning that they work well for the intendedinteraction, while others are non-preferred, meaning that they workpoorly for the interaction. Thus, in one embodiment, the non-preferredresidues can be substituted. For example, the valine at the C-terminusis generally a preferred anchor residue because it produces a stronginteraction with the HLA molecule and, thus, it was less preferred tosubstitute this residue. However, modifications of wild-typetumor-associated peptide epitopes by incorporating favorable anchorresidues have generated analogs with improved binding profiles with HLAmolecules and enhanced immunogenicity. One of the most successfulexamples is the A27L peptide analog of Melan-A 26-35 epitope (Valmori D,et al. J. Immunol. 160(4): 1750-8, 1998; which is hereby incorporated byreference in its entirety). The original epitope failed to form a stablecomplex with HLA-A2 molecules since it lacked an optimal anchor residueat position 2. In contrast the modified Melan A₂₆₋₃₅ A27L peptide analogdemonstrated unequivocally increased binding profiles with HLA-A2molecules and greater immunogenicity than its wild-type counterpart.Immunizing patients with this analog generated strong T cell immuneresponses that were able to recognize the wild-type epitope presented atthe cell surfaces. Similar modifications were obtained successfully withmany other tumor-associated epitopes such as GP100 209-217 (Parkhurst MR, et al. J. Immunol. 157(6): 2539-48, 1996; which is herebyincorporated by reference in its entirety), Her-2369-377 (Vertuani S, etal. J. Immunol. 172(6): 3501-8, 2004; which is hereby incorporated byreference in its entirety).

The choice of how many residues to substitute involves a desire tosubstitute better residues while still retaining enough of the qualitiesof the epitope that it will still be recognized by T cells whichrecognize the wild-type epitope. Thus, in one embodiment, one or twosubstitutions can be made to the wild-type peptide. In a furtherembodiment, more than two substitutions can be made to the wild-typepeptide, while still retaining cross-reactivity with the wild-typepeptide.

Thus, generally, the part of the peptide that is involved in TCRrecognition is desirably substituted to produce improved immunogenicitywhile still cross-reacting with the wild-type epitope. For example, apeptide that shows increased immunogenicity is preferred. Because the P2or second amino acid at the N-terminal end is believed to be primarilyinvolved in the process of producing improved immunogenicity, primarilythrough improved binding properties, it is a preferred substitution siteand a number of modifications were made in the exemplary analogs toidentify desired substitutions. Similar considerations apply to thecarboxy-terminal position, PΩ which also can be important for MHCbinding.

Thus, in one embodiment, the analog can include a substitution at the P2residue that substitutes a more hydrophobic residue for the wild-typealanine. In a further embodiment, the hydrophobic residue also canpossess a more bulky side chain. In a further embodiment, the residue atP1 can be substituted with a more hydrophobic residue. In a furtherembodiment, residues P1 and P2 both can be substituted with morehydrophobic residues. In further embodiments at least one residue at P1,P2, and P9 can be substituted. In a further embodiment, at least tworesidues at P1, P2 and P9 can be substituted. In a further embodiment atleast two residues at P1, P2, P9 P4 and P6 can be substituted includingone or more residues involved in TCR binding.

Further, substitutions of those residues only secondarily involved inbinding to TCR or the MHC molecule can be advantageous. For example,substitution of secondary TCR binding amino acids can generate analogsthat still bind and produce a response and do not interfere with thebinding to the MHC molecule, but preferably overcome the toleranceissues of self-antigens. This is useful because a patient who has cancermay be partially tolerized to the antigen. Thus, in order to overcomethat tolerance, an analog that retains some activity can be preferableto an analog with more improved immunogenicity, because it will be lesslikely to be recognized as “self” by the immune system.

1. N-Terminal Proximal Primary Anchor Modification (P2)

The N-terminal primary anchor is the 2^(nd) N-terminal amino acid of thepeptide and is the N-terminal proximal primary anchor. It is primarilyinvolved in the interaction with the MHC molecule and substitutions canresult in improved binding and stability. However, it may be secondarilyinvolved in TCR interactions also. Thus, substitutions at this site canresult in a peptide with improved interaction with MHC molecules as wellas improved interaction with the TCR.

The alanine found at this position in the wild-type sequence isgenerally believed to be non-preferred for the interaction with the MHCmolecule. Thus, preferred embodiments of the analogs have a substitutionat this position. In one embodiment, the original Ala 42 found in thewild-type sequence can be substituted with a more hydrophobic aminoacid. Any more hydrophobic amino acid may be used including any that isavailable or known to one of skill in the art, including standard aminoacids and non-standard amino acids. In a further embodiment, theoriginal Ala 42 is substituted with a more hydrophobic amino acid alsopossessing a bulky side chain. Examples of more hydrophobic amino acidsincludes, but are not limited to: Leu, Val, Ile, Met, α-aminobutyricacid, Norleucine and Norvaline.

TABLE 1 N-TERMINAL PROXIMAL PRIMARY ANCHOR MODIFICATION Cross-reactivity Half- and Predictive maximal Relative Stability fct avidityScores Binding affinity (T1/2) (native to SEQ ID Category Peptide nameSequence (R/NIH) (mM) (1/RA) (Hrs) analogs)* NO. Native SSX2 41-49KASEKIFYV 22/1017  14.64 1.0 11 1 1 peptide N-terminal SSX2 41-49KLSEKIFYV 28/73228 8.89 1.6 19 0.03 2 Primary (A42L) Anchor SSX2 41-49KVSEKIFYV 22/6407  5.2 2.8 20 0.03 3 (A42V) SSX2 41-49 KISEKIFYV26/10068 8.8 1.7 22.5 3 4 (A42I) SSX2 41-49 KMSEKIFYV 26/52887 8.8 1.722.5 0.1 5 (A42M) SSX2 41-49 K(D- NA N/B N/B N/B 10 6 (A42(D-Ala))Ala)SEKIFYV SSX2 41-49 K(D- NA N/B N/B N/B N/T 7 (A42(D-Leu))Leu)SEKIFYV SSX2 41-49 K(D- NA N/B N/B N/B 3 8 (A42(D-Val)) Val)SEKIFYVSSX2 41-49 KNaI- NA N/B N/B N/B >10 9 (A42(Nal-1)) 1SEKIFYV SSX2 41-49KNaI- NA 13.9 1.1 N/A 3 10 (A42(Nal-2)) 2SEKIFYV SSX2 41-49 KAbuSEKIFYVNA 7.56 1.9 N/A 0.3 11 (A42(Abu)) SSX2 41-49 KNIeSEKIFYV NA 5.82 2.5 240.1 12 (A42(Nle)) SSX2 41-49 KNvaSEKIFYV NA 11.4 1.3 N/A 0.1 13(A42(Nva)) SSX2 41-49 KAibSEKIFYV NA 18.4 0.8 N/A 3 14 (A42(Aib))

2. N-Terminal Secondary Anchor Modification (P1)

The N-terminal secondary anchor is the first amino acid at theN-terminus. This residue is Lys 41 and is defined as a secondary anchorresidue in interacting with the HLA-A*0201 molecule. However, it is alsoengaged in the interaction with the T cell receptors to a certaindegree. Therefore, modifications of this position can generate someheteroclitic analogs that are more immunogenic and more suitable for thedevelopment of tumor vaccines. Although the lysine at this position isgenerally considered to be favored, substitutions can result in highlyimproved properties.

Thus, in one embodiment, the original Lys 43 found in the wild-typesequence can be substituted with a more hydrophobic amino acid. Any morehydrophobic amino acid can be used, including any that is available orknown to one of skill in the art, including standard amino acids andnon-standard amino acids. In a further embodiment, the Lys 43 can besubstituted with an aromatic amino acid. Examples of more hydrophobicamino acids include, but are not limited to: Phe, Tyr, Trp, and D-Lys.

TABLE 2 N-TERMINAL SECONDARY ANCHOR MODIFICATIONS Cross- reactivityPredic- Half- and fct tive maximal Relative Stability avidity SEQ ScoresBinding affinity (T1/2) (native to ID Category Peptide name Sequence(R/NIH) (mM) (1/RA) (Hrs) analogs)* NO. Native SSX2 41-49 KASEKIFYV22/1017 14.64 1.0 11 1 1 N-terminal SSX2 41-49 FASEKIFYV 23/1336 9.551.5 >24 0.3 15 Secondary (K41F) Anchor SSX2 41-49 WASEKIFYV 22/133627.07 0.5 N/A >10 16 (K41W) SSX2 41-49 YASEKIFYV 21/1336 8.74 1.7 >24 317 (K41Y) SSX2 41- (D-Lys)ASEKIFYV NA N/B N/B N/B >10 18 49(K41(D-Lys))SSX2 41-49 PhgASEKIFYV NA 5.83 2.5 >24 0.1 19 (K41(Phg)) SSX2 41-49ChaASEKIFYV NA N/B N/B N/B >10 20 (K41(Cha)) SSX2 41-49 Phe(4-F)ASEKIFYVNA 6.72 2.2 >24 3 21 (K41(Phe-4F)) SSX2 41-49 Phe(4-NO2)ASEKIFYV NA 12.81.1 N/A 3 22 (K41(Phe-4NO2)) SSX2 41-49 O-methyl-TyrASEKIFYV NA 19.5 0.820 3 23 (K41(O-methyl Tyr)) SSX2 41-49 b-(3- NA 24.1 0.6 N/A 10 24(K41(b-(3- benzothienyl) benzothienyl)Ala)) AlaASEKIFYV

3. N-Terminal Primary and Secondary Modifications (P2 and P1)

In one embodiment, both primary and secondary anchor residues weresubstituted to result in improved binding affinity to the HLA molecule.In a further embodiment, the double substitution produced improvedstability of binding to the HLA molecule. In further embodiments, thebinding and/or stability was not improved and may have even beenreduced, but other properties of the molecule were improved, such asactivity or recognition by a tolerized individual.

TABLE 3 N-TERMINAL PRIMARY AND SECONDARY ANCHOR MODIFICATION Cross-reactivity Predic- Half- and fct tive maximal Relative Stability aviditySEQ Scores Binding affinity (T1/2) (native to ID Category Peptide nameSequence (R/NIH) (mM) (1/RA) (Hrs) analogs)* NO. Native SSX2 41-49KASEKIFYV 22/1017  14.64 1.0 11 1  1 N-terminal SSX2 41-49 (K41Y,YLSEKIFYV 29/96243 11.8 1.2 >24 N/T 25 Primary/ A42L) Secondary AnchorSSX2 41-49 (K41Y, YVSEKIFYV 23/8421   14.6 1.0 >24 0.1 26 A42V)SSX2 41-49 (K41Y, YMSEKIFYV 27/69508 25 0.6 >24 3 27 A42M)SSX2 41-49 (K41Y, YISEKIFYV 27/13233 6.5 2.3 N/A 1 28 A42I)SSX2 41-49 (K41F, FLSEKIFYV 28/96243 4.9 3.0 >24 0.3 29 A42L)SSX2 41-49 (K41F, FVSEKIFYV 22/8421  4.675 3.1 24 0.1 30 A42V)SSX2 41-49 (K41F, FMSEKIFYV 26/69508 6.58 2.2 >24 3 31 A42M)SSX2 41-49 (K41F, FISEKIFYV 26/13233 5.368 2.7 >24 0.3 32 A421)SSX2 41-49 (K41W, WLSEKIFYV 27/96243 4.472 3.3 >24 0.3 33 A42L)SSX2 41-49 (K41W, WVSEKIFYV 21/8421  4.82 3.0 >24 1 34 A42V)SSX2 41-49 (K41W, WMSEKIFYV 25/69508 5.13 2.9 >24 1 35 A42M)SSX2 41-49 (K41W, WISEKIFYV 25/13233 6.98 2.1 >24 0.1 36 A421)SSX2 41-49 (D-Lys) N/A 2.5 5.9 15 10 37 (K41(D-Lys), A42L) LSEKIFYVSSX2 41-49 (D- N/A 24.5 0.6 N/A 10 38 (K41(D-Lys), A42V) Lys) VSEKIFYV

4. N-Terminal Primary/Secondary Anchor and C-Terminal PrimaryModification (P2, P1 and P9)

The C-terminal Val of the wild-type peptide is generally a preferredanchor residue and primarily involved in the interaction with the MHCmolecule. However, substitutions were carried out to identify whichamino acids improve the analogs having primary and secondary N-terminalmodifications. These C-terminal substitutions can be used in the absenceof one or more N-terminal modifications also.

These modifications were shown to improve binding affinity and stabilityand in some cases resulted in analogs with decreased cross-reactivity.Thus, in some embodiments, the substitution to the C-terminus resultedin a peptide with improved binding and/or stability without decreasedcross-reactivity. However, in other embodiments the substitution to theC-terminus resulted in a peptide with improved binding and/or stabilitywith equal or decreased cross-reactivity. Each of the molecules can beof use in certain cases or in certain patients. In one embodiment, thevaline at the C-terminus is substituted with a large aliphatic aminoacid.

TABLE 4 N-TERMINAL PRIMARY/SECONDARY ANCHOR ANDC-TERMINAL PRIMARY MODIFICATIONS Cross- reactivity Predic- Half- and fcttive maximal Relative Stability avidity SEQ Scores Binding affinity(T1/2) (native to ID Category Peptide name Sequence (R/NIH) (M) (1/RA)(Hrs) analogs)* NO. Native SSX2 41-49 KASEKIFYV 22/1017  14.64 1.0 11 1 1 N-terminal SSX2 41-49 (K41F, FVSEKIFYL 22/2586  10.7 1.4 17 >10 39Primary/ A42V, V49L) Secondary Anchor, C-terminal Primary AnchorSSX2 41-49 (K41F, FVSEKIFYI 20/1263 9 1.6 24 0.3 40 A42V, V49I)SSX2 41-49 (K41F, FVSEKIFYA 16/601   6.9 2.1 16 1 41 A42V, V49A)SSX2 41-49 (K41F, FVSEKIFYM 16/601   17.8 0.8 22 >10 42 A42V, V49M)SSX2 41-49 (K41F, FVSEKIFY(Nle) N/A 5.59 2.6 >24 >10 43 A42V, V49Nle)SSX2 41-49 (K41F, FVSEKIFY(Nva) N/A 1.89 7.7 20 0.1 44 A42V, V49Nva)SSX2 41-49 (K41F, FVSEKIFY N/A 17.9 0.8 22 10 45 A42V, V49MeVal) (MeVal)SSX2 41-49 (K41F, FVSEKIFY N/A N/A N/A N/A >10 47 A42V, V49Aib) (Aib)SSX2 41-49 (K41F, FVSEKIFY N/A 3.43 4.3 20 1 48 A42V, V49Abu) (Abu)N-terminal SSX2 41-49 (A42V, KVSEKIFYI 20/961   13.9 1.1 N/A 0.3 70Primary V49I) Anchor, C- terminal Primary Anchor SSX2 41-49 (A42L,KLSEKIFYI 26/10984 5.682 2.6 N/A 0.03 71 V491) SSX2 41-49 (A42a, K(D-N/A N/B N/B N/B >10 72 V49v) Ala)SEKIF Y(D-Val) C-terminalSSX2 41-49 (V49I) KASEKIFYI 20/ 14 1.0 N/A 10 75 Primary 152.56 Anchor

5. N-Terminal Primary/Secondary Anchor and TCR Residues Modification

The TCR sites are generally recognized as residues P4, P6, and P8 andare the primary residues involved in the binding to the TCR. However,other residues may also be involved in the interaction to a lesserextent. In one embodiment, one or more of the sites primarily involvedin TCR interaction can be substituted to increase the interaction.Preferably, these substitutions can generate heteroclitic analogs thatdo not interfere with binding to the MHC molecule, but overcome thetolerance issues of the wild-type peptides. In a further embodiment, atleast one TCR substitution can be included with at least onesubstitution at position P1, P2, and/or P9. In a further embodiment, thesubstitution at any one or more of the P4, P6, and P8 positions can be apolar amino acid. In a further embodiment, the substitution can be anaromatic amino acid at position P8. In a further embodiment, thesubstitution can be an amino acid with a large aliphatic side chain atposition P6. In a further embodiment, the substitution can be an aminoacid which has a larger side chain to preserve the interaction.

TABLE 5 N-TERMINAL PRIMARY/SECONDARY ANCHOR AND TCR SITES MODIFICATIONCross- reactivity Predic- Half- and fct tive maximal Relative Stabilityavidity SEQ Scores Binding affinity (T1/2) (native to ID CategoryPeptide name Sequence (R/NIH) (mM) (1/RA) (Hrs) analogs)* NO. NativeSSX2 41-49 KASEKIFYV 22/1017 14.64 1.0 11 1  1 N-terminalSSX2 41-49 (K41F, FVSDKIFYV 21/8421 13.18 1.1 N/A >10 49 Primary/A42V, E44D) Secondary Anchor, TCR sites SSX2 41-49 (K41F, FVSNKIFYV20/2054 8.97 1.6 N/A >10 50 A42V, E44N) SSX2 41-49 (K41F, FVSSKIFYV20/2054 17.5 0.8 N/A >10 51 A42V, E44S) SSX2 41-49 (K41F, FVSTKIFYV20/2054 12.94 1.1 N/A >10 52 A42V, E44T) SSX2 41-49 (K41F, FVSQKIFYV20/2054 40.8 0.4 N/A 10 53 A42V, E44Q) SSX2 41-49 (K41F, FVS(Nle) N/A 131.1 N/A 10 54 A42V, E44Nle) KIFYV SSX2 41-49 (K41F, FVS(Nva) N/A 3.83.9 >24 3 55 A42V, E44Nva) KIFYV SSX2 41-49 (K41F, FVSEKLF 22/8421 7.81.9 24 3 56 A42V, I46L) YV SSX2 41-49 (K41F, FVSEKVF 22/8421 N/A N/A 241 57 A42V, I46V) YV SSX2 41-49 (K41F, FVSEKMF 18/8421 9.2 1.6 22 >10 58A42V, I46M) YV SSX2 41-49 (K41F, FVSEK(NI N/A 12.8 1.1 19 10 59A42V, I46NIe) e)FYV SSX2 41-49 (K41F, FVSEK(N N/A 6.21 2.4 >24 1 60A42V, I46Nva) va)FYV SSX2 41-49 (K41F, FVSEKIFTV 24/1531 3.9 3.8 24 >1061 A42V, Y48T) SSX2 41-49 (K41F, FVSEKIFF 22/8421 8.8 1.7 20 10 62A42V, Y48F) V SSX2 41-49 (K41F, FVSEKIF 24/1531 3.8 3.9 20 >10 63A42V, Y48S) SV SSX2 41-49 (K41F, FVSEKIF N/A 10.6 1.4 24 10 64A42V, Y48(Phe-4F)) (Phe-4F)V SSX2 41-49 (K41F, FVSEKIF N/A 5.852.5 >24 >10 65 A42V, Y48Phg) (Phg)V SSX2 41-49 (K41F, FVSEKLF 24/15315.67 2.6 24 >10 66 A42V, I46L, Y48T) TV SSX2 41-49 (K41F, FVSEKLF24/1531 N/A N/A N/A N/T 67 A42V, I46L, Y48S) SV N-terminalSSX2 41-49 (K41F, FVSEKLF 18/109  6.3 2.3 12 >10 68 Primary/A42V, I46L, Y48T, TA Secondary V49A) Anchor, C-terminal PrimaryAnchor, TCR sites SSX2 41-49 (K41F, FVSEKLF 18/109  6.2 2.4 N/A >10 69A42V, I46L, Y48S, SA V49A)

6. C-Terminal Amide

In some embodiments, the C-terminal residue can be modified to containan amide in the place of the free carboxylic acid. Thus, for example, ifthe peptide is a 9-mer (nonamer) the P9 residue can be modified. If thepeptide is a 10-mer (decamer) the P10 residue can be modified.Preferably this results in a peptide or analog that has increasedstability in biological media, including but not limited to blood,lymph, and CNS. Preferably, the peptides can retain the other necessaryactivities to result in an analog usable for vaccination or as animmunogen.

TABLE 6  C-TERMINAL AMIDE Cross- Predic- Half- reactivity tive maximalRelative Stability and fct avidity SEQ Scores Binding affinity (T1/2)(native to ID Category Peptide name Sequence (R/NIH) (M) (1/RA) (Hrs)analogs)* NO. Native SSX2 41-49 KASEKIFYV 22/1017 14.64 1.0 11 1  1 C-SSX2 41-49- KASEKIFYV- N/A N/B N/B N/T >10 76 terminal NH2 NH2 amideSSX2 41-49- KLSEKIFYV- N/A N/B N/B N/T 3 77 NH2 (A42L) NH2 SSX2 41-49-KVSEKIFYV- N/A N/B N/B N/T 10 78 NH2 (A42V) NH2

7. Decamers

The length of typical MHC binding peptides can vary from about 8 toabout 11 amino acids in length. However, most of the previously usedHLA-A*0201 are 9-mers (nonamers) or 10-mers (decamers). Thus, in oneembodiment, the analog can be an analog of the wild-type sequenceSSX-2₄₁₋₅₀. However, because the wild-type 10-mer does not have thecorrect binding motif and showed no immunological activity, a 10-mer wascreated by substituting amino acids at the P10 position and identifyingthe effect of various wild-type and analogs (see FIG. 1).

8. Remaining Residues

With reference to FIGS. 1A and 1B, any residues can also be substitutedwith conservative amino acids. Conservative substitutions can be pairedwith any of the above substitutions that can produce an effect.Alternatively, conservative substitutions can be specifically atresidues that are not believed to be involved in any of the activitiesat a primary, secondary, or even tertiary level. Such residues includeP3, P5 and P7. For example, the Serine at position P3 can be substitutedwith an alanine or threonine to produce an analog. Typically, suchconservative substitutions do not significantly affect the activity ofthe analog, however, in some embodiments they can increase certainactivities or decrease certain activities.

NY-ESO-1₁₅₇₋₁₆₅ Analogs

Many features regarding a variety of embodiments and aspects of analogdesign are disclosed above, either generally or as applied to the SSX-2epitope. It is to be understood that such disclosure is also applicableto this and subsequent epitopes. Explicit restatement of such disclosurewill be minimized for the sake of brevity.

Embodiments relate to analogs of the MHC class I-restricted T cellepitope NY-ESO-1₁₅₇₋₁₆₅, SLLMWITQC (SEQ ID NO. 98), polypeptidescomprising these analogs that can be processed by pAPC to present theepitope analogs, and nucleic acids that express the analogs. The analogscan have similar or improved immunological properties compared to thewild-type epitope.

One embodiment relates to methods to derivatize and improve analogs ofNY-ESO-1₁₅₇₋₁₆₅, along with specific sequences that encompasssubstitutions. The analogs can contain at least one substitution, butcan have multiple substitutions comprising standard or non-standardamino acids singly or in various combinations. The analogs can result inpeptides with retained or improved properties.

The epitope NY-ESO-1₁₅₇₋₁₆₅ has been shown to be presented by NY-ESO-1expressing cell lines, by measuring the epitope specific T cell activityagainst such cells (Jaeger, E. et al., J. Exp. Med. 187:265-270, 1998;U.S. Pat. No. 6,274,145 entitled ISOLATED NUCLEIC ACID MOLECULE ENCODINGCANCER ASSOCIATED ANTIGEN, THE ANTIGEN ITSELF, AND USES THEREOF), whichis incorporated herein by reference in its entirety. Methodologies toimprove the physico-chemical properties of the peptide NY-ESO-1₁₅₇₋₁₆₅have been described (U.S. Pat. No. 6,417,165 entitled NY-ESO-1-PEPTIDEDERIVATIVES, AND USES THEREOF), which is incorporated herein byreference in its entirety, and can consist of replacement of theterminal cysteine with other amino acids that preserve or enhance theinteraction with MHC and are devoid of the deleterious property ofdisulfide C—C bond formation interfering with the activity. However,sole manipulation of the C terminal cysteine residue ignores theadvantages of optimizing multiple residues throughout the peptide formajor histocompatibility (MHC) and/or T cell Receptor (TCR) binding.Thus, beyond the practicality of mutating the Cys residue, there isconsiderable opportunity in mutating additional amino acids throughoutthe peptide. For example, substitutions can be used to further optimizethe binding to MHC and/or TCR in a fashion that enables more effectiveapplication in clinics.

Embodiments relate to families of one or more peptides of 9 or 10 aminoacids in length related by sequence to amino acids 157-165 of the humancancer testis (CT) antigen NY-ESO-1 (NY-ESO-1₁₅₇₋₁₆₅).

Analog Design

The analog is generally an analog of the NY-ESO-1₁₅₇₋₁₆₅, with thesequence SLLMWITQC (SEQ ID NO. 98). Analysis of whether wild-type aminoacids are preferred or non-preferred used previous analyses of otherpeptide-MHC or TCR interactions. For example, the Cysteine at theC-terminus is generally a non-preferred anchor residue because it doesnot produce a strong interaction with the HLA molecule and, thus, it washighly preferred to substitute this residue. However, although theSerine at position P1 is generally preferred, it was found thatsubstituting an aromatic could produce a peptide with improvedproperties. Further the Leucine at position P2 is generally acceptable,but substituting a hydrophobic and/or bulky amino acid resulted in apeptide with improved properties. The residues which are primarilyinvolved in the interaction with the TCR (P4, P6 and P8) showed apreference generally for some polarity, and in the case of P8 anaromatic generally produced peptides with favorable properties.

One preferred embodiment relates to an analog that has a substitution atthe P2 position. Preferably, the substitution can be a hydrophobicresidue. More preferably, the substitution can be a bulky hydrophobicresidue. In a further embodiment, the residue at P1 can be substitutedwith a more hydrophobic residue. In a further embodiment, residues P1and P2 can be both substituted with more hydrophobic residues. Infurther embodiments at least one residue at P1, P2, and P9 can besubstituted. In a further embodiment, at least two residues at P1, P2and P9 can be substituted. In a further embodiment at least two residuesat P1, P2, P9, P4, and P6 can be substituted, including one or moreresidues involved in TCR binding. In a further embodiment, the residueat P8 can be substituted with an aromatic. Examples of the followingsubstitutions are shown in FIGS. 13A-13C.

1. N-Terminal Proximal Primary Anchor Modification (P2)

The N-terminal primary anchor is the 2^(nd) N-terminal amino acid of thepeptide, thus, it is the N-terminal proximal primary anchor. Althoughthe original Leucine 158 is not considered “non-preferred” for bindingto the MHC molecule, substitutions can produce a peptide with improvedbinding. Thus, in one embodiment, the original Leu 158 found in thewild-type sequence can be substituted with a similarly or morehydrophobic amino acid. Any hydrophobic amino acid may be used,including one that is available to or that is known to one of skill inthe art, including standard amino acids and non-standard amino acids. Ina further embodiment, the original Leu 158 can be substituted with amore hydrophobic amino acid also possessing a bulky side chain. Examplesof more hydrophobic amino acids include, but are not limited to: Leu,Val, Ile, Met, α-aminobutyric acid, Norleucine and Norvaline. Further, anaphthal side chain can also be substituted. Preferably, thesubstitution results in improved binding and stability with the HLAmolecule. However, this residue may be secondarily or tertiarilyinvolved in TCR interactions, and substitutions may also result inimproved recognition by the TCR.

2. N-Terminal Secondary Anchor Modification (P1)

The N-terminal secondary anchor is the first amino acid at theN-terminus or P1. This residue is involved in a number of interactions.The residue of Ser 157 was defined as a secondary anchor residue ininteracting with HLA-A*0201 molecule, it also engaged in the interactionwith the T cell receptors to a certain degree. Therefore, modificationsof this position generate some heteroclitic analogs that are moreimmunogenic and more suitable for the development of tumor vaccines.Thus, substitutions can result in a variety of improved qualities.

Although the Serine is not considered “non-preferred,” a number ofsubstitutions can result in improved qualities of the peptide. Thus, inone embodiment, the original Ser 157 found in the wild-type sequence canbe substituted with a more hydrophobic amino acid. Any more hydrophobicamino acid can be used, including one that is available to or that isknown to one of skill in the art, including standard amino acids andnon-standard amino acids. Examples of more hydrophobic amino acidsinclude, but are not limited to: Phe, Tyr, Tip, and D-Lys.

3. N-Terminal Primary and Secondary Modifications (P2 and P1)

In one embodiment, both primary and secondary anchor residues weresubstituted to result in improved binding affinity to the HLA molecule.In a further embodiment, the double substitution produced improvedstability of binding to the HLA molecule. In further embodiments, thebinding and/or stability was not improved and may have even beenreduced, but other properties of the molecule were improved, such asactivity or recognition by a tolerized individual.

4. N-Terminal Primary/Secondary Anchor and C-Terminal PrimaryModification (P2, P1 and P9)

The C-terminal cysteine of the wild-type peptide is generally anon-preferred anchor residue. Because this residue is generallyprimarily involved in the interaction with the MHC molecule, it can bepreferred to substitute residues that result in a stronger interactionwith the MHC molecule. Thus, substitutions were shown to improve bindingaffinity and stability and in some cases resulted in analogs withdecreased cross-reactivity. In some embodiments, the substitution to theC-terminus can result in a peptide with improved binding and/orstability without decreased cross-reactivity. However, in otherembodiments the substitution to the C-terminus can result in a peptidewith improved binding and/or stability with equal or decreasedcross-reactivity. Because substitution of this residue have beenpreviously shown to provide improved peptides, it can be preferableproduce peptides that were more improved in the interaction with the MHCmolecule as well as other interactions, such as the recognition by theTCR. Thus, in some embodiments, the C-terminal substitution can bepaired with at least one other substitution. Examples of amino acidsubstitutions to the C-terminus include, but are not limited to, valine,lysine, alanine, and isoleucine.

5. N-Terminal Primary/Secondary Anchor and TCR Residue Modifications

The primary residues involved in the interaction with the TCR aregenerally recognized as residues P4, P6, and P8. However, other residuesmay also be involved in the interaction to a lesser extent. In oneembodiment, one or more of the sites primarily involved in TCRinteraction can be substituted to result in an improved interaction.Preferably, these substitutions generate heteroclitic analogs that donot interfere with binding to the MHC molecule, but overcome thetolerance issues of the wild-type peptides. In one embodiment, at leastone TCR substitution can be included with at least one substitution atposition P1, P2, and/or P9. In one embodiment, amino acids with somepolarity can be substituted at P4, P6, and P8. In a further embodiment,amino acids which are aromatic can be substituted at the P8 position.

6. C-Terminal Amide

In some embodiments, the C-terminal residue can be modified to containan amide in the place of the free carboxylic acid. Thus, for example ifthe peptide is a 9-mer (nonamer) the P9 residue can be modified. If thepeptide is a 10-mer (decamer) the P10 residue can be modified.Preferably this results in a peptide or analog that has increasedstability in biological media, including but not limited to blood,lymph, and CNS. Preferably, the peptides retain the other activities toresult in an analog usable for vaccination or as an immunogen.

7. Decamers

The length of typical MHC binding peptides varies from about 8 to about11 amino acids in length. However, most of the previously usedHLA-A*0201 are 9-mers (nonamers) or 10-mers (decamers). Thus, in oneembodiment, the analog can be a 10-mer of the wild-type sequenceNY-ESO-1₁₅₇₋₁₆₆. However, because the wild-type 10-mer does not have thecorrect binding motif and showed no immunological activity, a 10-mer wascreated by substituting amino acids at the P10 position and identifyingthe effect of various wild-type and analogs (see FIGS. 13A-13C). In oneembodiment, the residues that were added or substituted for thewild-type at the C-terminus can be selected from the group consisting ofnorvaline, leucine, isoleucine, valine, and alanine.

8. Remaining Residues

With reference to FIGS. 13A and 13C, any residues can also besubstituted with conservative amino acids. Conservative substitutionscan be paired with any of the above substitutions that can produce aneffect. Alternatively, conservative substitutions can be specifically atresidues that are not believed to be involved in any of the activitiesat a primary, secondary, or even tertiary level. Such residues caninclude P3, P5 and/or P7. Conservative substitutions are known to thoseof skill in the art, but, for example, the Leucine at position P3 can besubstituted with an alanine or threonine to produce an analog.Typically, such conservative substitutions do not significantly affectthe activity of the analog. However, in some embodiments they mayincrease certain activities or decrease certain activities. Because ofthe known interactions, it is unlikely that such conservativesubstitutions will have a significant effect on any of the activities.

PSMA₂₈₈₋₂₉₇ Analogs

Many features regarding the variety of embodiments and aspects of analogdesign are disclosed above, either generally or as applied to particularepitopes. It is to be understood that such disclosure is also applicableto this and subsequent epitopes. Explicit restatement of such disclosurewill be minimized for the sake of brevity.

Some embodiments relate to analogs of the MHC class I-restricted T cellepitope PSMA₂₈₈₋₂₉₇, GLPSIPVHPI (SEQ ID NO. 114), polypeptidescomprising these analogs that can be processed by pAPC to present theepitope analogs, and nucleic acids that express the analogs. The analogscan have similar or improved immunological properties compared to thewild-type epitope. Evidence validating the presentation of this epitopeby human cancer cells is presented in Example 32 below.

One embodiment relates to methods to derivatize and improve analogs ofPSMA₂₈₈₋₂₉₇, along with specific sequences that encompass substitutions.The analogs can contain at least one substitution, but can have multiplesubstitutions comprising standard or non-standard amino acids singly orin various combinations. The analogs may result in peptides withretained or improved properties.

Embodiments relate to families of one or more peptides of 9 or 10 aminoacids in length related by sequence to amino acids 288-297 of the humanPSMA.

Analog Design

In some embodiments, the PSMA₂₈₈₋₂₉₇ analog can contain substitutions ofthe sequence GLPSIPVHPI (SEQ ID NO. 114). Reference to binding motifdata, such as presented in table 7 in example 2 below, indicates thatthe P2 anchor residue can make the largest individual contribution toaffinity of any position in an A2.1-restricted epitope. In this case theamino acid at the P2 position is the optimally preferred leucine. The PΩanchor residue, isoleucine, is favorable. In vitro binding studies usingthe T2 cell assay system (not shown) have indicated that the nativepeptide has generally superior binding characteristics, particularly ascompared to the SSX-2 and NY-ESO-1 epitopes. The epitope exhibitedsignificant binding at relatively low concentrations, although this waspaired with a relatively shallow rise toward saturation. The wild-typeepitope can be improved. Analyses such as that represented by tables 7and 8 are averages and the behavior of a given residue in a particularsequence may diverge from the average. Consistent with the favorableresults obtained with Nle and Nva for the SSX-2 and NY-ESO-1 epitopesdiscussed above, Nle and Nva also can be successfully used for theinstant PSMA epitope. Finally, even similar binding characteristics, ifpaired with alterations that help circumvent whatever tolerance to theepitope may exist, can increase the effective immunogenicity of thepeptide. In the transgenic mouse model the native peptide is poorlyimmunogenic (see Example 35 for instance) which may reflect tolerance tothe epitope; the region of PSMA from which this epitope is derived isidentical between mouse and human PSMA.

1. N-Terminus Proximal Primary Anchor Modification (P2)

As noted above, although the native residue at the P2 position of thisepitope is generally the optimal residue among genetically encoded aminoacids, the effect of substituting other preferred or bulky hydrophobicresidues were examined for potential improvement of binding, tolerancebreaking and cross-reactive immunity. Exemplary substitutions caninclude Met, Ile, Gln, Val, Nva, Nle, and aminobutyric acid (Abu).

2. N-Terminal Secondary Anchor Modification (P1)

The N-terminal secondary anchor is the first amino acid at theN-terminus. The native Gly is only marginally preferred at thisposition. Various observations (see tables 7 and 8 for example) showthat amino acids with potential to improve the epitope include Ala, Ser,Abu and sarkosine (Sar, that is, N-methylglycine).

3. C-Terminal Primary Anchor Modification (PΩ)

The native Ile at this position is generally a preferred but not optimalresidue. Substitution at this position can improve binding. Exemplarysubstitutions can include Val, Leu, Nva, and Nle.

4. Secondary Anchors and TCR Exploration

The penultimate position (PΩ−1) can serve both as a secondary anchor anda TCR interacting position. Substitution of Ala, Leu, Ser, and Thr canbe have their primary effect on TCR interaction, though they can alsocontribute to improved binding. P3 is another position that can effectboth binding and immunogenicity. Substitution of Trp at this positioncan improve both.

Further embodiments relate to combinations of substitutions at multiplepositions in order to combine, synergize, and counteract the variouseffects obtained with the single substitutions.

PRAME₄₂₅₋₄₃₃ Analogs

Many features regarding the variety of embodiments and aspects of analogdesign are disclosed above, either generally or as applied to particularepitopes. It is to be understood that such disclosure is also applicableto this and subsequent epitopes. Explicit restatement of such disclosurewill be minimized for the sake of brevity.

Embodiments include analogs of the MHC class I-restricted T cell epitopePRAME₄₂₅₋₄₃₃, SLLQHLIGL (SEQ ID NO. 115), polypeptides comprising theseanalogs that can be processed by pAPC to present the epitope analogs,and nucleic acids that express the analogs. The analogs can have similaror improved immunological properties compared to the wild-type epitope.Evidence validating the presentation of this epitope by human cancercells is presented in Example 39 below.

One embodiment relates to methods to derivatize and improve analogs ofPRAME₄₂₅₋₄₃₃, along with specific sequences that encompasssubstitutions. The analogs can contain at least one substitution, butcan have multiple substitutions comprising standard or non-standardamino acids singly or in various combinations. The analogs can result inpeptides with retained or improved properties.

Some embodiments relate to families of one or more peptides of 9 or 10amino acids in length related by sequence to amino acids 425-433 of thehuman PRAME sequence.

Analog Design

Some embodiments relate to analogs of the PRAME₄₂₅₋₄₃₃ which can containsubstitutions of the sequence SLLQHLIGL (SEQ ID NO. 115). Reference tobinding motif data, such as presented in table 7 in Example 2 below,indicates that the P2 anchor residue can make the largest individualcontribution to affinity of any position in an A2.1-restricted epitope.In this case the amino acid at the P2 position is the optimallypreferred leucine. The PΩ anchor residue, leucine, is favorable, thoughnot as strongly preferred. Analyses such as that represented by tables 7and 8 are averages and the behavior of a given residue in a particularsequence can diverge from the average, nor is the wild type PΩ residuenecessarily the most preferred for that position. Consistent with thefavorable results obtained with Nle and Nva for the other epitopes,similar improvements can be obtained substituting Nle and Nva with thissequence. Finally, even similar binding characteristics, if paired withalterations that help circumvent whatever tolerance to the epitope mayexist, can increase the effective immunogenicity of the peptide.

The rationale for various substitutions has been set forth above. Theparticular substitutions investigated for the PRAME₄₂₅₋₄₃₃ epitopefollow the same logic and are disclosed in the examples 40-42 and FIGS.25-27. Substitutions were made at the primary anchor positions P2 and PΩ(P9), the secondary anchor positions P1 and PΩ-1 (P8). Substitutionswere also made in the TCR interacting positions (in addition tosecondary anchor positions) P3 and P6. Selected substitutions haveimpact on binding and/or stability of MHC class I-peptide complexes, keyfeatures in determining the immunological properties of peptides. Inaddition, due to T cell repertoire considerations and to circumventmechanisms responsible for the limited immunity to native epitopes,substitutions that retain the capability of analogs to interact with Tcell receptors recognizing native peptides, can be of practical value.

EXAMPLES

The following examples provide analogs and methods of identifyinganalogs. The analogs can be used, for example, as immunogens, vaccines,and/or treatment of a variety of cancers. The analogs were produced asin Example 1. SSX-2₄₁₋₄₉ analogs were identified as shown in Example 2,those produced listed in Example 3 and tested for improved properties asin Examples 4-21. The testing of NY-ESO-1₁₅₇₋₁₆₅ analogs were tested forimproved properties as in Examples 22-30.

Example 1 Peptide Synthesis, Purification and Characterization

Peptides were synthesized on either a Symphony multiple peptidesynthesizer (PTI technologies, MA) or an ABI 433A peptide synthesizer(Applied Biosystems, Foster City, Calif.) at 0.05-0.1 mmole scale usingstandard Fmoc solid phase chemistry. C-terminal free acid peptides weresynthesized using pre-load PEG-PS resins (on Symphony) or Wang resin (onABI). C-terminal amidated peptides were synthesized on Fmoc-PAL-PEG-PSresin. All resins were purchased from Applied Biosystems (Foster City,Calif.). The Fmoc-amino acids used in peptide syntheses were purchasedfrom Novabiochem (San Diego, Calif.) and AnaSpec (San Jose, Calif.).Post-synthesis cleavage was carried on by the standard protocol.

Peptide purification was carried out on either semi-preparative HPLCcolumns or SPE cartridges (Phenomenex, Torrance, Calif.). The purity ofall peptides was ≧90%. The identity of each peptide was verified byMaldi-TOF MS (Voyager DE, Applied Biosystems) and analytical HPLCs(Varian or Shimazu) using a Synergi C12 column (Phenomenex, Torrance,Calif.).

Example 2 De Novo Designed SSX-241-49 Analogs

Structural modification of a moderately antigenic peptide canconsiderably improve peptide-MHC binding, CTL recognition, and/orimmunogenicity. General guidelines regarding how to modify a wild-typeepitope in order to achieve a peptide analog with enhanced potency areknown in the art. An appreciated strategy is to optimize the residues atthe so-called anchor positions for binding to the particular MHCmolecule at issue. In the case of HLA-A2 a marked preference forhydrophobic residues at the P2 and PΩ positions has been observed,particularly L, and M at P2, and V at PΩ. (PΩ denotes the C-terminalresidue of the epitope. For HLA-A2 that is P9 or P10 depending on thelength of the peptide.) Replacing the P1 position with aromaticresidues, such as F, Y and W can also be advantageous.

TABLE 7 Coefficients used by the BIMAS algorithm (Algorithm available byhypertext transfer protocol: //bimas.cit.nih.gov/molbio/hla_bind/) 9-merCoefficient Table for HLA_A_0201 Amino Acid Position Type 1st 2nd 3rd4th 5th 6th 7th 8th 9th A 1.000 1.000 1.000 1.000 1.000 1.000 1.0001.000 1.000 C 1.000 0.470 1.000 1.000 1.000 1.000 1.000 1.000 1.000 D0.075 0.100 0.400 4.100 1.000 1.000 0.490 1.000 0.003 E 0.075 1.4000.064 4.100 1.000 1.000 0.490 1.000 0.003 F 4.600 0.050 3.700 1.0003.800 1.900 5.800 5.500 0.015 G 1.000 0.470 1.000 1.000 1.000 1.0000.130 1.000 0.015 H 0.034 0.050 1.000 1.000 1.000 1.000 1.000 1.0000.015 I 1.700 9.900 1.000 1.000 1.000 2.300 1.000 0.410 2.100 K 3.5000.100 0.035 1.000 1.000 1.000 1.000 1.000 0.003 L 1.700 72.000 3.7001.000 1.000 2.300 1.000 1.000 4.300 M 1.700 52.000 3.700 1.000 1.0002.300 1.000 1.000 1.000 N 1.000 0.470 1.000 1.000 1.000 1.000 1.0001.000 0.015 P 0.022 0.470 1.000 1.000 1.000 1.000 1.000 1.000 0.003 Q1.000 7.300 1.000 1.000 1.000 1.000 1.000 1.000 0.003 R 1.000 0.0100.076 1.000 1.000 1.000 0.200 1.000 0.003 S 1.000 0.470 1.000 1.0001.000 1.000 1.000 1.000 0.015 T 1.000 1.000 1.000 1.000 1.000 1.0001.000 1.000 1.500 V 1.700 6.300 1.000 1.000 1.000 2.300 1.000 0.41014.000 W 4.600 0.010 8.300 1.000 1.000 1.700 7.500 5.500 0.015 Y 4.6000.010 3.200 1.000 1.000 1.500 1.000 5.500 0.015 final 0.069 constant

TABLE 8 Scoring Pattern for HLA-A*0201 used by the SYFPEITHI Algorithm(9-mers) (Algorithm available by hypertext transfer protocol://syfpeithi.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm) AA P1 P2P3 P4 P5 P6 P7 P8 P9 A 2 6 2 0 0 0 2 1 6 C 0 0 0 0 0 0 0 0 0 D −1 0 0 10 0 0 0 0 E −1 0 −1 2 0 0 0 2 0 F 1 0 1 −1 1 0 0 0 0 G 1 0 0 2 2 0 0 1 0H 0 0 0 0 0 0 1 0 0 I 2 8 2 0 0 6 0 0 8 K 1 0 −1 0 1 0 −1 2 0 L 2 10 2 01 6 1 0 10 M 0 8 1 0 0 0 0 0 6 N 0 0 1 0 0 0 1 0 0 P 0 0 0 2 1 0 1 0 0 Q0 0 0 0 0 0 0 0 0 R 0 0 0 0 0 0 0 0 0 S 2 0 0 0 0 0 0 2 0 T 0 6 −1 0 0 20 2 6 V 1 6 0 0 0 6 2 0 10 W 0 0 1 0 0 0 0 0 0 X 0 0 0 0 0 0 0 0 0 Y 2 01 −1 1 0 1 0 0 Adapted from: Rammensee, Bachmann, Stevanovic: MHCligands and peptide motifs. Landes Bioscience 1997

Example 3

The following analogs were produced using the predictions in Example 1.

TABLE 9 SEQ ID Catergory Number Peptide name Sequence wild-type  1SSX-2 41-49 KASEKIFYV N-terminal  2 SSX-2 41-49 (A42L) KLSEKIFYVPrimary Anchor  3 SSX-2 41-49 (A42V) KVSEKIFYV  4 SSX-2 41-49 (A42I)KISEKIFYV  5 SSX-2 41-49 (A42M) KMSEKIFYV  6 SSX-2 41-49 (A42(D-Ala))K(D-Ala)SEKIFYV  7 SSX-2 41-49 (A42(D-Leu)) K(D-Leu)SEKIFYV  8SSX-2 41-49 (A42(D-Val)) K(D-Val)SEKIFYV  9 SSX-2 41-49 (A42(Nal-1))KNal-1SEKIFYV 10 SSX-2 41-49 (A42(Nal-2)) KNal-2SEKIFYV 11SSX-2 41-49 (A42(Abu)) KAbuSEKIFYV 12 SSX-2 41-49 (A42(Nle)) KNleSEKIFYV13 SSX-2 41-49 (A42(Nva)) KNvaSEKIFYV 14 SSX-2 41-49 (A42(Aib))KAibSEKIFYV N-terminal 15 SSX-2 41-49 (K41F) FASEKIFYV Secondary Anchor16 SSX-2 41-49 (K41W) WASEKIFYV 17 SSX-2 41-49 (K41Y) YASEKIFYV 18SSX-2 41-49 (K41(D-Lys)) (D-Lys)ASEKIFYV 19 SSX-2 41-49 (K41(Phg))PhgASEKIFYV 20 SSX-2 41-49 (K41(Cha)) ChaASEKIFYV 21SSX-2 41-49 (K41(Phe-4F)) Phe(4-F)ASEKIFYV 22SSX-2 41-49 (K41(Phe-4NO2)) Phe(4-NO ₂)ASEKIFYV 23SSX-2 41-49 (K41(O-methyl Tyr)) O-methyl-TyrASEKIFYV 24SSX-2 41-49 (K41(β-(3-benzothienyl)A1a)) β-(3-benzothienyl) AlaASEKIFYVN-terminal 25 SSX-2 41-49 (K41Y, A42L) YLSEKIFYV Primary/SecondaryAnchor 26 SSX-2 41-49 (K41Y, A42V) YVSEKIFYV 27 SSX-2 41-49 (K41Y, A42M)YMSEKIFYV 28 SSX-2 41-49 (K41Y, A42I) YISEKIFYV 29SSX-2 41-49 (K41F, A42L) FLSEKIFYV 30 SSX-2 41-49 (K41F, A42V) FVSEKIFYV31 SSX-2 41-49 (K41F, A42M) FMSEKIFYV 32 SSX-2 41-49 (K41F, A42I)FISEKIFYV 33 SSX-2 41-49 (K41W, A42L) WLSEKIFYV 34SSX-2 41-49 (K41W, A42V) WVSEKIFYV 35 SSX-2 41-49 (K41W, A42M) WMSEKIFYV36 SSX-2 41-49 (K41W, A42I) WISEKIFYV 37 SSX-2 41-49 (K41(D-Lys), A42L)(D-Lys)LSEKIFYV 38 SSX-2 41-49 (K41(D-Lys), A42V) (D-Lys)VSEKIFYVN-terminal 39 SSX-2 41-49 (K41F, A42V, V49L) FVSEKIFYL Primary/SecondaryAnchor, C-terminal Primary Anchor 40 SSX-2 41-49 (K41F, A42V, V49I)FVSEKIFYI 41 SSX-2 41-49 (K41F, A42V, V49A) FVSEKIFYA 42SSX-2 41-49 (K41F, A42V, V49M) FVSEKIFYM 43SSX-2 41-49 (K41F, A42V, V49Nle) FVSEKIFY(Nle) 44SSX-2 41-49 (K41F, A42V, V49Nva) FVSEKIFY(Nva) 45SSX-2 41-49 (K41F, A42V, V49MeVal) FVSEKIFY(MeVal) 46SSX-2 41-49 (K41F, A42V, V49MeLeu) FVSEKIFY(MeLeu) 47SSX-2 41-49 (K41F, A42V, V49Aib) FVSEKIFY(Aib) 48SSX-2 41-49 (K41F, A42V, V49Abu) FVSEKIFY(Abu) N-terminal 49SSX-2 41-49 (K41F, A42V, E44D) FVSDKIFYV Primary/SecondaryAnchor, TCR sites  50 SSX-2 41-49 (K41F, A42V, E44N) FVSNKIFYV 51SSX-2 41-49 (K41F, A42V, E44S) FVSSKIFYV 52SSX-2 41-49 (K41F, A42V, E44T) FVSTKIFYV 53SSX-2 41-49 (K41F, A42V, E44Q) FVSQKIFYV 54SSX-2 41-49 (K41F, A42V, E44(Nle)) FVS(Nle)KIFYV 55SSX-2 41-49 (K41F, A42V, E44(Nva)) FVS(Nva)KIFYV 56SSX-2 41-49 (K41F, A42V, I46L) FVSEKLFYV 57SSX-2 41-49 (K41F, A42V, I46V) FVSEKVFYV 58SSX-2 41-49 (K41F, A42V, I46M) FVSEKMFYV 59SSX-2 41-49 (K41F, A42V, I46(Nle)) FVSEK(Nle)FYV 60SSX-2 41-49 (K41F, A42V, I46(Nva)) FVSEK(Nva)FYV 61SSX-2 41-49 (K41F, A42V, Y48T) FVSEKIFTV 62SSX-2 41-49 (K41F, A42V, Y48F) FVSEKIFFV 63SSX-2 41-49 (K41F, A42V, Y48S) FVSEKIFSV 64SSX-2 41-49 (K41F, A42V, Y48(Phe-4F)) FVSEKIF(Phe4-F)V 65SSX-2 41-49 (K41F, A42V, Y48(Phg)) FVSEKIF(Phg)V 66SSX-2 41-49 (K41F, A42V, I46L, Y48T) FVSEKLFTV 67SSX-2 41-49 (K41F, A42V, I46L, Y48S) FVSEKLFSV N-terminal 68SSX-2 41-49 (K41F, A42V, I46L, Y48T, FVSEKLFTA Primary/Secondary V49A)Anchor, C-terminal  Primary Anchor, TCR sites 69SSX-2 41-49 (K41F, A42V, I46L, Y48S, FVSEKLFSA V49A) N-terminal   70SSX-2 41-49(A42V, V49I) KVSEKIFYI Primary Anchor, C-terminalPrimary Anchor 71 SSX-2 41-49(A42L, V49I) KLSEKIFYI 72SSX-2 41-49(A42(D-Ala), V49(D-Val)) K(D-Ala)SEKIFY(D-Val) 73SSX-2 41-49(A42(D-Leu), V49(D-Val)) K(D-Leu)SEKIFY(D-Val) 74SSX-2 41-49(A42(D-Val), V49(D-Val)) K(D-Val)SEKIFY(D-Val)C-terminal Primary 75 SSX-2 41-49(V49I) KASEKIFYI AnchorC-terminal amide 76 SSX-2 41-49-NH2 KASEKIFYV-NH2 77SSX-2 41-49-NH2(A42L) KLSEKIFYV-NH2 78 SSX-2 41-49-NH2(A42V)KVSEKIFYV-NH2 Decamers 79 SSX-2 41-50 KASEKIFYVY 80 SSX-2 41-50(Y50I)KASEKIFYVI 81 SSX-2 41-50(Y50L) KASEKIFYVL 82 SSX-2 41-50(Y50V)KASEKIFYVV 83 SSX-2 41-50(Y50(Nle)) KASEKIFYV(Nle) 84SSX-2 41-50(Y50(Nva)) KASEKIFYV(Nva) 85 SSX-2 41-50(A42V, Y50I)KVSEKIFYVI 86 SSX-2 41-50(A42L, Y50I) KLSEKIFYVI 87SSX-2 41-50(A42V, Y50L) KVSEKIFYVL 88 SSX-2 41-50(A42L, Y50L) KLSEKIFYVL89 SSX-2 41-50(A42V, Y50V) KVSEKIFYVV 90 SSX-2 41-50(A42L, Y50V)KLSEKIFYVV 91 SSX-2 41-50(A42V, Y50(Nle)) KVSEKIFYV(Nle) 92SSX-2 41-50(A42L, Y50(Nle)) KLSEKIFYV(Nle) 93SSX-2 41-50(A42V, Y50(Nva)) KVSEKIFYV(Nva) 94SSX-2 41-50(A42L, Y50(Nva)) KLSEKIFYV(Nva) 95SSX-2 41-50(A42V, V49I, Y50I) KVSEKIFYII 96SSX-2 41-50(A42L, V49I, Y50I) KLSEKIFYII 97 SSX-2 41-50(V49I, Y50I)KASEKIFYII Abbreviations for non-standard amino acids: Nle, norleucine;Nva, norvaline; Phg, phenylglycine; Phe(4-F), 4-fluorophenylalanine;Phe(4-NO₂), 4-nitrophenylalanine; Abu, α-aminobutyric acid; Aib,α-aminoisobutyric acid; MeLeu, methyl-leucine; MeVal, methylvaline;β-(3-benzothienyl)Ala, β-(3-benzothienyl)-alanine; O-methyl-Tyr,O-methyltyorosine; Cha, cyclohexylalanine; Nal-1, β-(1-napthyl)-alanine;Nal-2, β-(2-napthyl)-alanine; —NH2 indicates that the carboxy terminushas been modified to the amide.

Examples 4-21 Testing of SSX-2₄₁₋₄₉ Analogs

The analogs produced in Example 3 were tested for activity, such asbinding and biological effect as follows in Examples 4-21:

Example 4 Peptide Binding Using T2 Cells

The affinity of peptide analogs and the wild-type epitope to HLA-A*0201was evaluated using a T2 cell based assay (Regner M, et al., Exp ClinImmunogenet. 1996; 13(1):30-5; which is hereby incorporated by referencein its entirety).

For the binding assay, in brief, the T2 cells that lack expression ofTAP and thus do not assemble stable MHC class I on the cell surface,were pulsed with different concentrations of peptides (controls oranalogs) overnight at 37° C., washed extensively, stained withfluorescently tagged antibody recognizing MHC class I (A2 allele) andrun through a FacsScan analyzer. The difference between the MFI (meanfluorescence intensity) corresponding to a given concentration of analogand the negative control (non-MHC binder) is a function of how manystabilized complexes between MHC and peptide are displayed on thesurface of T2 cells. Thus, at limiting concentrations of peptide, thisis a measurement of K_(on) mostly and at saturation levels of peptidethat is a measurement of both K_(on) and K_(off). The binding wasquantified by two factors that are mathematically related: half maximalbinding (the peptide concentration giving 50% of the signalcorresponding to saturation) and relative affinity (1/RA). Relativeaffinity RA is binding normalized to a reference (wild-type peptide);for example, the ratio between half max binding of control relative topeptide analog. The higher the 1/RA index and the lower the half maximalbinding, the higher the K_(on) of the interaction between the analog andthe MHC. Fifty three analogs were identified with these bindingparameters improved relative to the wild-type peptide. These improvedbinders carry one, two, three or multiple substitutions (includingstandard and/or non-standard amino acids) involving positions that areknown to participate in the interaction with MHC and/or TCR. However,the overall effect on MHC binding was dependent on the modification.Such peptide analogs can be useful in therapeutic compositions or as aplatform to further derive therapeutic compositions.

Example 5 Peptide Stability Using T2 Cells

Peptide stability (K_(off)) on MHC generally cannot be solely inferredfrom binding (K_(on)). In addition, along with binding, the stability ofpeptides on MHC class I is notoriously important in regard to theimmunological properties of such peptides, since the activation of Tcells depends on the duration of “signal 1” (MHC peptide complexinteraction with T cell receptor). For the stability assay, in brief,the T2 cells that lack expression of TAP and thus do not assemble stableMHC class I on the cell surface, were pulsed with a concentration ofpeptide (controls or analogs) known to achieve maximal loading of MHCclass I (“saturation”) overnight at 37° C., washed extensively, andchased for different intervals in the presence of emetine, which blocksendogenous protein synthesis. After extensive washing, the cells werestained with fluorescently tagged antibody recognizing MHC class I (A2allele) and run through a FacsScan analyzer. The difference between theMFI (mean fluorescence intensity) corresponding to a given concentrationof analog and the negative control (non-MHC binder) is a function of howmany stabilized complexes between MHC and peptide are displayed on thesurface of T2 cells. The decay of the signal over time wasmathematically expressed as stability index 50% relative to the bindingat 0 hours (at the beginning of the chase interval).

Such improved analogs can carry one, two, three or multiplesubstitutions (including standard and/or non-standard amino acids)involving positions that are known to participate in the interactionwith MHC and/or TCR, with an overall effect on MHC stability that isdependent on the modification. Such peptide analogs can be useful intherapeutic compositions or as a platform to further derive therapeuticcompositions. Forty three of the analogs have increased stabilityrelative to the natural peptide.

The analogs that showed both improved binding and stability are usefulin improved compositions or as a platform to generate improvedcompositions of therapeutic benefit.

Example 6 Evaluation of Immunologic Properties of AnalogsCross-Reactivity and Functional Avidity

The immunologic properties of peptides can be described as a function ofbinding to MHC molecules (K_(on) and K_(off)) and TCR (affinity ofinteraction between TCR and MHC-peptide complexes). Modifications ofprimary MHC anchor residues generally have a significant degree ofpredictability in regard to overall impact on binding to MHC molecules.

Modifications of secondary MHC anchor residues may impact the affinityof interaction of the MHC-peptide complex to TCR along with the K_(on)and K_(off) relative to peptide-MHC interaction.

A methodology was devised that allowed rapid and rational screening ofpeptide analogs in a fashion coherent with proposed methods of use andmodeling the overall immunologic properties (K_(on) and K_(off) relativeto MHC interaction and TCR binding properties in an integrated fashion).This method can include generating T cell lines against a natural(non-mutated) epitope (SSX-2₄₁₋₄₉) using an immunization strategy potentenough to generate a useful response in transgenic mice carrying humanMHC (such as the A2 allele). Peptide analogs were interrogated ex vivoin the presence of competent APCs and the functional impact of T cellsspecific for natural (non-mutated) epitope measured. The evaluation wasdone at various concentrations of analog, since the expected effect wasbiphasic in the case of cross reactive peptides (activating at limitedconcentrations and inhibiting at higher concentrations, due toantigen-induced cell death, AICD). Measurement of the following threeparameters can define basic and useful characteristics of peptideanalogs:

-   -   1. Minimal required concentration of peptide analog to trigger        effects indicative of T cell activation (e.g. cytokine        production);    -   2. Maximal (peak value) effect (e.g. cytokine production) at any        analog concentration;    -   3. Analog concentration at peak value of activating effect        (e.g., cytokine concentration)

For example, analogs that result in reduced values associated withparameters #1 and 3 but increased #2, can be useful. Use of naturalepitope and unrelated non-cross reactive peptides as references isvaluable in identifying classes of analogs of potential value. Analogsthat display properties quantitatively comparable to or even modestlyattenuated from those of natural epitopes are still deemed useful inlight of the fact that while they retain cross-reactivity, they maydisplay immunologic properties that are distinct from those of thenatural peptide—for example lower propensity to induce AICD or abilityto break tolerance or restore responsiveness in vivo.

Some advantages of this screening strategy include the practicality andrapidity, use of more relevant polyclonal T cell lines instead ofpotentially biased T cell clones as a read out, and the composite value,integrating parameters such as K_(on), K_(off) and TCR affinity that maytranslate into cross-reactivity and functional avidity of peptide-MHCcomplexes relative to TCR. These parameters can be predictive of the invivo immunologic properties and thus can delineate useful panels ofpeptide analogs to undergo further evaluation, optimization andpractical applications. Analogs that bind to MHC and retaincross-reactivity against TCR specific for the nominal wild-type peptideare predicted to trigger a measurable effect in this assay. The overallmethodology is presented in FIG. 2.

The method used for the generation of T cell lines was the following:HHD transgenic mice carrying an A2 human allele (Pascolo et al. J. ExpMed. 185(12):2043-51, 1997, which is hereby incorporated herein byreference in its entirety) were immunized with 50 ug of SSX-2 naturalepitope (41-49) admixed with 25 ug of pIpC at day 0, 4, 14 and 18 bybilateral administration into the inguinal lymph nodes. At 7 days afterthe last boost, the mice were sacrificed and a suspension of splenocytesprepared at 5×10⁶ million cells/ml in complete HL-1 medium. Cells wereincubated with different concentrations of peptide for 48 hours inflat-bottomed 96-well plates (200 ul/well) and for an additional 24hours with rIL-2 at 10 U/ml added to the wells. The supernatant washarvested and the concentration of IFN-gamma assessed by standardmethods such as ELISA.

Example 7 Cross-Reactivity and Functional Avidity of Analogs Substitutedat Single Position

The strategy from above (Example 6, FIG. 2) was applied to scan througha library of analogs bearing single substitutions relative to thenatural SSX-2₄₁₋₄₉ epitope (KASEKIFYV (SEQ ID NO. 1)) in its wild-typeversion (FIG. 3). Strong inverse correlation was found between theminimal required amount of analog to elicit IFN-gamma production ex vivoand the maximal amount of cytokine production at any concentration ofanalog.

Substitution of A₄₂ with L, V or M improved on the immunologicproperties of the peptide, assessed in this assay. L and V mutants wereactive. M was more active than the natural epitope. The I mutantretained cross-reactivity to the TCR recognizing the wild-type epitope.

Replacement of the A at position 42 with non-standard amino acids Abu,Nle or Nva improved on the immunologic properties of the peptiderelative to the wild-type epitope, both in terms of the minimal amountof analog required to trigger cytokine production and the peak amount ofcytokine produced. Mutants encompassing D-Ala, D-Val, NaI-2 or Aibdisplay retained cross-reactivity and reduced immune activity in thisassay relative to the natural peptide, but can still be useful forfurther derivitization to adjust or enhance their properties. An NaI-1at position 42 abrogated the activity.

Changes of the first residue K₄₁ showed that, while replacement with For Phg improved on the activity, W, D-Lys, and Cha obliterated theimmunologic properties in this assay. Replacement of K with Y, Phe-4F,Phe(4-NO2), O-methyl-Tyr or beta-(3-benzothienyl-Ala) retained activity.

Modification of position V₄₉ (C-terminal residue) by replacement with Iretained the activity at a lower level compared to the original epitope.Modification of the last residue by addition of an —NH₂ moietyobliterated the activity of the peptide that was subsequently rescued bymodifying the A at position 42 with L or V. This illustrates directlythat analogs with activity that is lower than that of the wild-typepeptide are still useful for further derivatization.

Example 8 Cross-Reactivity and Functional Avidity of Analogs Substitutedat Two Positions

The strategy from above (Example 6, FIG. 2) has been applied to scanthrough a library of analogs bearing two substitutions, relative to thewild-type SSX-2₄₁₋₄₉ epitope in its wild-type version (FIG. 4).

Coordinated modifications at position 1 and 2 have a variable effect onthe activity of analogs. For example, substitution of K41 with Y, F or Wcorroborated with substitution of A42 with V, M or I, and resulted inpreserved or enhanced activity of the analogs relative to the wild-typepeptide. Such doubly mutated peptides offer an increased opportunity toimpact the interaction with TCR in a fashion that results in tolerancebreaking (thus being useful for practical application), since the P1residue participates to a certain extent in binding to TCR. Combinationsbetween the following: Y (position 41) with V (at position 42), W(position 41) with I or I (at position 42), and F (position 41) with L,V, I (at position 42) resulted in analogs that were more active relativeto the wild-type peptide. Combinations between Y at position 41 and I atposition 42, or W at position 41 and V or M at 42, conferred an activitysimilar to that of wild-type peptide. Replacement of K with D-lysine atposition 41 reduced resulted in analogs with retained activity in thisassay. Such peptides can be very useful since the metabolic degradationof such peptides encompassing non-standard amino acids is decreased invivo.

Combinations between V or L at position 42 and I at position 49 resultedin increased activity over the natural peptide.

Example 9 Cross-Reactivity and Functional Avidity of Analogs Substitutedat Multiple Positions

The strategy from above (Example 6, FIG. 2) has been applied to scanthrough a library of analogs bearing three or more substitutionsrelative to the natural SSX-2₄₁₋₄₉ epitope in its wild-type version(FIG. 5).

F and V at positions 41 and 42 respectively, combined with I or A atposition 49 resulted in improved or similar activity relative to thewild-type epitope. In contrast, L or M at position 49 resulted inheavily diminished activity.

Triple mutants comprising the non-standard amino acids Nva, Abu or MeValat the last position resulted in retention or improvement of immuneactivity. Such peptides are extremely useful due to increased in vivostability and resistance to enzymatic degradation.

Modification of amino acid residues within the putative TCR bindingregion can result in peptides of considerable value, that retain bindingto MHC along with cross-reactivity and thus be useful for restoration ofimmune responsiveness or tolerance breaking since their conformation inthe MHC groove is slightly different from that of natural peptides.Additional substitutions at position 44 (Q, Nva or Nle), position 46 (L,V, Nle or Nva) or 48 (F or Phe-4F) resulted in active analogs, whereasD, N, S or T at position 44, M at 46 or T, S, Phg at position 48 or L atposition 46 with T at 48, resulted in analogs devoid of activity.Finally, two analogs with 5 substitutions showed no activity (FIG. 5).

Example 10 Cross-Reactivity and Functional Avidity of DecamersEncompassing The Natural Peptide and Mutated at Various Positions

The strategy from above (Example 6, FIG. 2) has been applied to scanthrough a library of analogs of a decamer encompassing the nominalSSX-2₄₁₋₄₉ peptide (FIG. 6).

The decamer SSX-2 41-50 was significantly less active in stimulating theT cell line specific for the 41-49 nonamer, relative to the latter.Modification of the Y residue at position 50 to I or L, but to a lesseror no extent to V, Nle or Nva, resulted in restoration of activity inthis assay. Further optimization of the activity of decameric analogscan be obtained by modification of the A at position 2 with L or V. TheA42L substitution rescued the activity of the Y50Nva decamer. Peptideanalogs of similar or reduced activity in vitro (but retainedcross-reactivity) compared with the natural peptide are still useful forinduction or boost of immune responses due to: i) more limited AICD; ii)potentially higher in vivo activity due to increased stability on classI MHC and/or slightly modified interaction with TCR which is can beimportant for tolerance breaking.

Example 11 Use of Analogs to Trigger Enhanced Immunity Against NaturalEpitope, Assessed Ex Vivo

Three groups of mice (n=4) were immunized with a plasmid expressingSSX-2₄₁₋₄₉ natural epitope, by direct inoculation into the inguinallymph nodes with 25 ug in 25 ul of PBS/each lymph node at day 0, 3, 14and 17. This was followed by two additional peptide boosts (similaramount) at day 28 and 31. The schedule of immunization is shown in FIG.7. One week after the boost, splenocytes were stimulated ex vivo withSSX-2₄₁₋₄₉ natural peptide and tested against ⁵¹Cr-labeled target cells(T2 cells) at various E:T ratios (FIG. 8). The results showed that theanalog A42V triggered a higher response against target cells expressingthe natural peptide, compared to the analog A42L or the wild-typepeptide itself, as boost agents. This correlated with the binding andstability parameters determined by ex vivo experimentation.

Example 12 Use of Analogs to Trigger Enhanced Immunity Against NaturalEpitope, Assessed In Vivo

Eight groups of mice (n=4) were immunized with plasmid expressingSSX-2₄₁₋₄₉ natural epitope, by direct inoculation into the inguinallymph nodes with 25 ug in 25 ul of PBS/each lymph node at day 0, 3, 14and 17. This was followed by two additional peptide boosts (similaramount) at day 28 and 31, using a negative control peptide (Melan A26-35 “EAA”), natural peptide or analogs as shown in FIG. 9.

To evaluate the in vivo response against natural peptide, splenocyteswere isolated from littermate control HHD mice and incubated with 20μg/mL or 1 ug/ml of natural peptide for 2 hours. These cells were thenstained with CFSE^(hi) fluorescence (4.0 μM or 1 μM for 15 minutes) andintravenously co-injected into immunized mice with an equal number ofcontrol splenocytes stained with CFSE^(lo) fluorescence (0.4 μM).Eighteen hours later the specific elimination of target cells wasmeasured by removing spleen and PBMC from challenged animals andmeasuring CFSE fluorescence by flow cytometry. The relative depletion ofthe populations corresponding to peptide loaded splenocytes wascalculated relative to the control (unloaded) population and expressedas % specific lysis. FIG. 10 (spleen) and 11 (blood) show the in vivocytotoxicity elicited by the regimens described in FIG. 7. Three of thetested peptides (A42V, K41F and K41Y) showed increased activity relativeto the natural peptide, both in spleen and blood, against target cellscoated with 20 as well as 1 ug/ml of natural peptide. Interestingly,there was only limited correlation between binding, stability of analogsin regard to the interaction with MHC, and the capability to generate invivo immunity against natural peptide (FIG. 11).

Example 13 Use of Analogs to Trigger Enhanced Responses Against TumorCells

Eight groups of mice (n=4) were immunized with plasmid expressingSSX-2₄₁₋₄₉ natural epitope, by direct inoculation into the inguinallymph nodes with 25 ug in 25 ul of PBS/each lymph node at day 0, 3, 14and 17. This was followed by two additional peptide boosts (similaramount) at day 28 and 31, using a negative control peptide (Melan A26-35 “EAA”), natural peptide or analogs as shown in FIG. 9.

One week after the boost, splenocytes were stimulated ex vivo withSSX-2₄₁₋₄₉ wild-type peptide and tested against ⁵¹Cr-labeled human tumorcells (624.38 melanoma cells) at various E:T ratios (FIG. 12). AnalogA42V and K41F A42V V49I elicited immune responses that mediatedincreased cytotoxicity against human tumor cells expressing the naturalSSX-2₄₁₋₄₉ epitope.

Example 14 N-Terminal Proximal Primary Anchor Modification (2^(ND) AA)

When the substituted analogs shown in Table 3 were tested, the analogsshowed improved binding and stability profiles in comparison with thewild-type peptide epitope. However, the magnitude of improvement foreach analog varied, and the substitution of A42V showed the highestimprovement in terms of binding affinity with HLA-A*0201 molecule.Further, the stability of the A42V-HLA-A*0201 complex was better thanthe complex formed between wild-type peptide and HLA-A*0201: the T1/2extended from 11.5 hrs to 20 hrs. The peptides with 42 A to L, V and Msubstitutions were able to induce the IFN-γ secretion of wild-typepeptide specific CTL at remarkable lower concentrations. The 42A to Isubstitution generated an analog with improved binding and stabilityprofile. The residue at the P2 position can also be engaged in theinteraction with TCR to a certain degree. This observation was alsosupported by the results with the 42 A to Aib analog, which possessed asimilar binding affinity with HLA-A*0201 relative to the wild-typeepitope.

Example 15 N-Terminal Secondary Anchor Modification (1^(ST) AA)

The N-terminal secondary anchor is the first amino acid at theN-terminus. Thus, in one embodiment, the original Lys 43 found in thewild-type sequence is substituted with a more hydrophobic and bulkyamino acid. Any more hydrophobic and bulky amino acid also can be used,including any available to or that is known to one of skill in the art,including standard amino acids and non-standard amino acids. Examples ofmore hydrophobic amino acids include, but are not limited to: Phe, Tyr,Trp, and D-Lys.

The residue of Lys 41 was defined as a secondary anchor residue ininteracting with HLA-A*0201 molecule, and it also engaged in theinteraction with the T cell receptors to a certain degree. Therefore,modifications of this position can generate some heteroclitic analogsthat are more immunogenic and more suitable for the development of tumorvaccines.

From Table 3, one could see that by replacing Lys 41 to Tyr, Phe or Phederivatives (Phenylglycine, Para-fluorophenylalanine,Para-nitrophenylalanine), the resulting analogs have higher affinitywith the HLA-A*0201 molecule and form more stable complexes. On theother hand, the Lys to Tip or Trp derivatives analogs have shownsignificantly decreased affinity with the HLA-A*0201 molecule althoughbased on the predicted algorithms, the Trp analog should have a similaraffinity to that of the Tyr and Phe analogs. The experimental data havedemonstrated the limitation of the predicted algorithms. For examples:Lys 41 to Phg substitution has resulted in an analog with improvedaffinity and extended stability with the HLA-A*0201 molecule, however,its cross-reactivity with wild-type peptide specific CTL was fairlypoor. On the other hand, the para-nitrophenylalanine analog was shown toinduce the IFN-γ secretion of the wild-type peptide specific CTL at amuch lower concentration, although its affinity with the HLA-A*0201molecule was about the same as that of wild-type peptide.

Example 16 N-Terminal Primary/Secondary Anchor Modification

When both primary and secondary anchor residues at the N-terminal weremodified, a general trend was that resulting analogs demonstratedimproved affinity and extended stability with the HLA-A*0201 molecule(Table 3), with only a few exceptions: (K41Y, A42V), (K41Y, A42M) and(K41(D-Lys), A42V). Additionally, they had very good cross-reactivitywith the wild-type peptide specific CTL. Combining the K41W substitutionwith A42V or A42L improved the binding/stability profile, these analogsand also had desirable cross-reactivity activity with the wild-typepeptide. The combination modifications of N-terminal primary anchor andsecondary anchor changed the peptide structure and conformation to agreater degree.

Example 17 N-Terminal Primary/Secondary Anchor and C-Terminal PrimaryModification

The C-terminal Val of the wild-type peptide was a preferred anchorresidue. However, improved potency was observed when it was mutated toIle, having one additional —CH₂ group; similar improvement was alsoobserved with a Val to Abu substitution. Although the other analogsshowed improved binding affinity and stability with the MHC molecule,their cross-reactivity results were poor. The results of these analogsindicated that the peptide C-terminal anchor residue also plays acritical role in the recognition of T cells. (Table 4).

Example 18 N-Terminal Primary/Secondary Anchor and TCR SitesModification

Substitutions of secondary TCR binding amino acid residues preferablygenerate heteroclitic analogs that did not interfere with the binding tothe MHC molecule, but overcame the tolerance issues of self-antigens. Bycombining the substitutions of N-terminal primary/secondary anchorresidues (K41F and A42V) and the TCR sites, analogs were generated withimproved binding affinity and stability (Table 6). Some of these analogsinduced the IFN-γ production of the wild-type peptide specific CTL atlower concentrations, such as K41F, A42V, E44(Nva)/(Nle) mutants andK41F, A42V, 146L/(Nva)/(Nle) mutants.

Example 19 N-Terminal Amide

Replacing the peptide's free carboxylic acid C-terminus with an amideimproved the peptide's stability in biological media by conferringstability to proteolysis and conferred dipeptidyl carboxypeptidaseresistance to the peptide. However, some of the resultant analogs lost asignificant amount of their affinity with MHC molecules, as well asimmunogenicity and antigenicity. Interestingly, although the threeanalogs (Table 7) disclosed in this application lost their bindingcapability with MHC molecule, SSX-2₄₁₋₄₉ —NH₂ (A42V) retained itsreactivity with wild-type peptide specific CTLs as indicated by itscapability of inducing the secretion of IFN-γ at a similar concentrationto that of the wild-type peptide. SSX-2₄₁₋₄₉ —NH₂ (A42L) was, however,able to stimulate the IFN-γ production at a lower concentration.

Example 20 Decamers

The length of typical MHC binding peptides varies from 8-mer to 11-mer,and most HLA-A*0201 binding peptides are 9-mers or 10-mers. In previousobservations, a 9-mer and 10-mer from a natural sequence were both foundto possess a binding motif for the same MHC, and had the sameN-terminus. From the standpoint of proteasomal processing they aredistinct epitopes, but were nonetheless antigenically cross-reactive. Inthe case of the wild-type epitope SSX-2₄₁₋₄₉, the epitope is a 9-merpeptide and the 10-mer peptide, SSX-2₄₁₋₅₀, lacks the appropriate MHCbinding motif and showed no immunological activity. Thus, the wild-typeepitope was therefore lengthened to a 10-mer with amino acids that couldcreate the appropriate binding motif. As shown in Table 8, while many10-mer analogs have a lower binding affinity with the HLA-A*0201molecule, analogs SSX-2₄₁₋₅₀ (A42L, Y50L/V/Nle/Nva) showed improvedbinding affinity with the HLA-A*0201 molecule. Two 10-mer analogs inparticular, A42L and Y50 Nle/Nva, were able to induce IFN-γ productionfrom T cells immunized against the wild-type peptide at lowerconcentrations than the wild-type peptide.

Example 21 Use of Analogs to Overcome Tolerization

One aspect in which the analogs can represent an improvement over thewild-type epitope is in increased immunogenicity in a human system andtolerance breaking Differences in the TCR repertoire, whether due togerm line differences or differences in negative selection, have thepotential to give anomalous results. To address such issues the analogsare used in an in vitro immunization of HLA-A2⁺ blood to generate CTL.Techniques for in vitro immunization, even using naive donors, are knownin the field, for example, Stauss et al., Proc. Natl. Acad. Sci. USA89:7871-7875, 1992; Salgaller et al. Cancer Res. 55:4972-4979, 1995;Tsai et al., J. Immunol. 158:1796-1802, 1997; and Chung et al., J.Immunother. 22:279-287, 1999; each of which is hereby incorporated byreference in their entirety.

Specifically, PBMCs from normal donors were purified by centrifugationin Ficoll-Hypaque from buffy coats. All cultures were carried out usingautologous plasma (AP) to avoid exposure to potential xenogeneicpathogens and recognition of FBS peptides. To favor the in vitrogeneration of peptide-specific CTL, autologous dendritic cells (DCs)were employed as APCs. DCs were generated and the CTLs were induced withDCs and peptides from PBMCs as described in Keogh et al., 2001, which isincorporated herein by reference in its entirety. Briefly,monocyte-enriched cell fractions were cultured for 5 days with GM-CSFand IL-4 and were cultured for 2 additional days in culture media with 2μg/ml CD40 ligand to induce maturation. 2×10⁶ CD8+-enriched Tlymphocytes/well and 2×10⁵ peptide-pulsed DCs/well were co-cultured in24-well plates in 2 ml RPMI supplemented with 10% AP, 10 ng/ml IL-7 and20 IU/ml IL-2. Cultures were restimulated on days 7 and 14 withautologous irradiated peptide-pulsed DCs. Immunogenicity was assayedusing the in vitro cytotoxicity and cytokine production assays describedherein.

Examples 22-30 Testing of NY-ESO-1₁₅₇₋₁₆₅ Analogs

The analogs listed in FIG. 13 were tested for activity, such as bindingand biological effect as follows in Examples 22-30:

Example 22 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at a Single Position (FIGS. 13 A-C)

The strategy from above (Example 6) was applied to scan through alibrary of analogs bearing single substitutions relative to thewild-type NY-ESO-1₁₅₇₋₁₆₅ epitope in its native (or wild-type) version(FIG. 13). Strong inverse correlation was found between the minimalrequired amount of analog to elicit IFN-gamma production ex vivo and themaximal amount of cytokine production at any concentration of analog.

Substitution of S157 with F or K, resulted in analogs that partiallyretained MHC binding and cross-reactivity with the T cells specific forthe nominal epitope. Substitution of L158 with I improved theimmunologic features of the peptide as assessed by this methodology;whereas L158V resulted in partial retention of activity. Modification ofC165 with any of the amino acids V, L, A, or I resulted in improvedimmune properties.

Peptides that have substitutions in the N-terminal position orelsewhere, and present with retained but not increased activity in thisassay relative to the wild-type peptide, can be useful in humans. Inaddition, they are material for further derivatization to improve ontheir properties, as described below.

Example 23 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Two Positions (FIG. 13A-L)

The strategy from above (Example 6) was applied to scan through alibrary of analogs bearing two substitutions relative to the wild-typeNY-ESO-1₁₅₇₋₁₆₅ epitope. Simultaneous semi-conservative modifications atposition 2 and 9 were shown to have profound effects on the immuneproperties of analogs, depending on the precise identity of the analogs.Combining L158I with C165V or C165L further increased their activityrelative to the wild-type peptide. Similarly, L158V improved on theactivity of the C165V or C165L analogs, further increasing such activityrelative to wild-type peptide. L158V partially retained the activity ofC165A or C1651 analogs, showing an interesting effect of double mutationof primary anchor residues. Similarly, L158I partially retained theactivity of the C165A analog.

Simultaneous modifications at positions 1 and 9 had profound effects onthe immune properties of analogs, depending on the precise identity ofthe analogs. S157Y combined with C165Nva (norvaline) or Nle (norleucine)at position 9 resulted in substantially improved activity over S157Yalone or the wild-type peptide. The C165V mutant also rescued theactivity of the S157Y mutant. V—NH₂ or L-NH₂ at position 9 partiallyrescued the activity of the S157Y analog—however, A-NH₂ failed to do so.Combinations between S157F and V, L, I, and to a lesser extent A at the9^(th) position retained strong activity of the analog and may be moreuseful than single mutants at position 9 due to the participation of thefirst residue in the interaction with TCR. Combinations between S157Kand V, L, I and to a lesser extent A at the 9^(th) position, retainedstrong activity of the analog and may be more useful than single mutantsat position 9 due to the participation of the first residue in theinteraction with TCR and the overall beneficial effect on the peptidesolubility of K at position 1.

Example 24 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Multiple Positions (FIG. 13A-L)

The strategy from above (Example 6) was applied to scan through alibrary of analogs bearing multiple substitutions relative to thewild-type NY-ESO-1 epitope.

L158Nva or L158Nle considerably improved on the activity of the S157YC165V mutant. Combinations between V or I at position 158 and V, L, A orI at 165 can partially restore the potency of analogs relative to thewild-type peptide. 5157Y L158I C165V displayed increased activityrelative to the wild-type peptide and S157V with C165V or C1651; andS1571 with C165L or I, retained MHC binding and cross-reactivity with Tcells specific for the wild-type peptide.

Triple substitutions comprising Y and V at positions 157 and 165,respectively, in addition to L or N at 160; A, L, V, or N at 162; or E,D or T at 164, retained the activity of the peptide in thiscross-reactivity assay, making these analogs useful compounds forbreaking T cell tolerance in vivo since positions 160, 162 and 164participate in the interaction with TCR.

Triple substitutions comprising 157F and 158V plus V, L, I at theposition 165 showed activity in the assay described in Example 2. Inaddition, triple mutants encompassing S157F and L158I plus V or A atposition 165 retained activity. Together, these data underline thecomplex interactive and non-linear nature of multiple substitutions.

Finally, triple mutants comprising S157W and to a higher extent S157Ttogether with 158V and 165V, showed retained or increased activityrespectively, relative to the wild-type peptide.

Example 25 Cross-Reactivity and Functional Avidity of DecamersEncompassing the Wild-Type Peptide and Mutated at Various Positions(FIG. 13A-L)

The strategy from above (Example 6) was applied to scan through alibrary of analogs of a decamer encompassing the nominal NY-ESO-1₁₅₇₋₁₆₅peptide. While the decamer itself lacked significant in vitro activity,various substitutions at this position partially rescued activity, suchas L at 166, or L, I, Nle at 166 combined with Y at 157 and V at 165.

Peptide analogs with similar or reduced activity in vitro (but withretained cross-reactivity) compared to the wild-type peptide are stilluseful for induction or boost of immune responses due to: i) morelimited AICD (antigen-induced cell death); ii) higher in vivo activitydue to increased stability on class I MHC and/or slightly modifiedinteraction with TCR. Thus, these analogs are useful for breakingtolerance.

Example 26 Evaluation of Immunologic Properties of Analogs PeptideBinding to MHC Class I Molecules (FIG. 13A-L)

The affinity of peptide analogs and the wild-type epitope to HLA-A*0201was evaluated by T2 cell based assay (ref. Regner M, et al., Exp ClinImmunogenet. 1996; 13(1):30-5), which is incorporated herein byreference in its entirety. For the binding assay, in brief, T2 cells,that lacked expression of TAP and thus do not assemble stable MHC classI on the cell surface, were pulsed with different concentrations ofpeptides (controls or analogs) overnight at 37° C., washed extensively,stained with fluorescently tagged antibody recognizing MHC class I (A2allele) and run through a FacsScan analyzer. Peptides that bind A2stabilize its presence at the cell surface. The difference between theMFI (mean fluorescence intensity) corresponding to a given concentrationof analog and the negative control (a non-MHC binding peptide) is afunction of how many stabilized complexes between MHC and peptide aredisplayed on the surface of T2 cells. Thus, at limiting concentrationsof peptide, this is a measurement of K_(on) mostly and at saturationlevels of peptide that is a measurement of both K_(on) and K_(off). InFIG. 13 the binding is quantified by two factors that are mathematicallyrelated: Half maximal binding (the peptide concentration giving 50% ofthe signal corresponding to saturation) and relative affinity (1/RA),that is binding normalized to a reference (wild-type peptide)—i.e. theratio between half maximal binding of control relative to peptideanalog. The higher the 1/RA index and the lower the half maximalbinding, the higher the K_(on) of the interaction between an analog andthe MHC molecules. In FIG. 13, there are 39 analogs described with suchbinding parameters improved relative to the wild-type peptide. Suchimproved binders carry one, two, three, or more substitutions ofstandard and/or non-standard amino acids at positions that are known toparticipate in the interaction with MHC and/or TCR, with an overalleffect on MHC binding that is dependent on precise/conjugatedmodification. Such peptide analogs are useful in therapeuticcompositions or as a platform to further derive therapeuticcompositions.

Example 27 Method of Immunization (FIG. 14)

Eight groups of mice (n=4) were immunized with a plasmid expressing thewild-type NY-ESO-1₁₅₇₋₁₆₅ epitope, by direct inoculation into theinguinal lymph nodes with 25 ug in 25 ul of PBS into each lymph node atdays 0, 3, 14 and 17. This was followed by two peptide boosts (similaramount) at day 28 and 31, using a negative control peptide (HBVc),wild-type peptide or analog as shown in FIG. 14.

Example 28 Use of Analogs to Trigger Enhanced Immunity Against Wild-TypeEpitope, Assessed In Vivo (FIGS. 15A-C)

To evaluate the in vivo responses obtained against the wild-typeepitope, splenocytes were isolated from littermate control HHD mice andincubated with 20 μg/mL or 1 μg/ml of wild-type peptide for 2 hours.These cells were then stained with CFSE^(hi) and CFSE^(med) fluorescence(4.0 μM or 1 μM, respectively, for 15 minutes) and intravenouslyco-injected into immunized mice with an equal number of controlsplenocytes stained with CFSE^(lo) fluorescence (0.4 μM). Eighteen hourslater the specific elimination of target cells was measured by removingthe spleens and PBMC from challenged animals and measuring CFSEfluorescence by flow cytometry. The relative depletion of thepopulations corresponding to peptide loaded splenocytes was calculatedrelative to the control (unloaded) population and expressed as %specific lysis. FIG. 15A shows the lack of in vivo cytotoxicity in micereceiving the negative control peptide. FIG. 15B shows the variablecytotoxicity in mice immunized with plasmid and amplified with wild-typepeptide. FIG. 15C shows the substantial, constant cytotoxicity in miceimmunized with plasmid and amplified with the analog L158Nva C165V.

Example 29 Comparison of Various Analogs in Triggering Enhanced ImmunityAgainst the Wild-Type Epitope, Assessed In Vivo (FIGS. 16A-B)

In the context of the immunization protocol described above (Example 8)and using the methodology described in the Example 9, in vivocytotoxicity against target cells coated with limited (1 μM; FIG. 16A)or supraoptimal amounts of wild-type peptide (20 μM, FIG. 16B) wasevaluated subsequent to the entrain and amplify protocol using plasmidand peptide analog respectively for the two stages. Results expressed asaverage % specific lysis+/−SE showed that the analog L158V C165Nvainduced the highest activity and that the analogs L158V C165V, L158VC165Nva and S157K L158V C165V showed an effect in the same range withwild-type peptide or the C165V mutant. Since multiple substitutions mayalter the TCR binding site, such analogs can be more useful than thewild-type peptide in breaking tolerance against a self epitope. Inaddition, the S 157K triple mutant can ameliorate the poor solubility ofthe wild-type peptide or other analogs, with direct practicalimplications.

Example 30 Use of Analogs to Trigger Enhanced Immunity Against theWild-Type Epitope, Assessed Ex Vivo by Cytokine Production (FIGS. 17A-B)

In the context of the immunization protocol described above (Example27), and following the challenge described in Example 28, splenocyteswere isolated, pooled and stimulated in vitro with 10 μM of wild-typepeptide NY-ESO-1₁₅₇₋₁₆₅ for 3 and 6 days respectively. Supernatants wereharvested and the concentration of IFN-γ measured by ELISA. AnalogL158Nva C165V induced T cells that produced large levels of IFN-gammamore rapidly upon ex vivo stimulation (FIG. 17A). Other analogs such asS157F L158V C165V, L158V C165Nva, and L158V C165V induced T cells thatproduced increased amounts of IFN-gamma upon ex vivo re-stimulation withwild-type peptide (FIG. 17B). In contrast, C165V failed to induceincreased capability of T cells to produce IFN-γ, relative to thewild-type peptide following the protocols described in Examples 27-28.

Example 31 Characterization of Binding and Stability by ELISA (ItopiaTesting)

Avidin-coated microtiter plates containing class I monomer loaded with aso-called placeholder peptide were used to evaluate peptide binding,affinity and off-rate. The monomer-coated plates were supplied as partof the iTopia Epitope Discovery System Kit (Beckman Ciulter, Inc., SanDiego, Calif., USA). Assay buffers, anti-MHC-FITC mAb andbeta2-microglobulin and control peptides were also supplied with thekits.

Binding Assay:

Native peptide and analogs were first evaluated for their ability tobind each MHC molecule by binding assay. This assay measures the abilityof individual peptides to bind HLA molecules under standardized optimalbinding conditions. Monomer-coated plates were first stripped, releasingthe placeholder peptide and leaving only the MHC heavy chain bound tothe plate. Test peptides were then introduced under optimal foldingconditions, along with the anti-MHC-FITC mAb. Plates were incubated for18 hrs at 21° C. The anti-MHC-FITC mAb binds preferentially to arefolded MHC complex. Therefore, the fluorescence intensity resultedfrom each peptide was related to the peptide's ability to form complexwith MHC molecule. Each peptide's binding was evaluated relative to thepositive control peptide provided in the kit, and the results wereexpressed ‘% binding’. The analogs identified as ‘better-binders’ inrelative to the native peptide were subsequently analyzed in theaffinity and off-rate assays.

Affinity Assay:

For the affinity assay, after the initial stripping of the placeholderpeptide, increasing concentrations (range 10⁻⁴ to 10⁻⁸ M) of each testpeptides for a given allele were added to a series of wells andincubated under the conditions described previously. Plates were read onthe fluorometer. Sigmoidal dose response curves were generated usingPrism software. The amount of peptide required to achieve 50% of themaximum was recorded as ED₅₀ value.

Off-Rate Assay:

For the off-rate assay, the plates were washed after 18 hrs incubationat 21° C. to remove the excess amount peptide. The plates were thenincubated on the allele-specific monomer plates at 37 C. The plates weremeasured at multiple time points (0, 0.5, 1, 1.5, 2, 4, 6 and 8 hrs) forrelative fluorescence intensity. The time required for 50% of thepeptide to dissociate from the MHC monomer is defined as the T1/2 value(hrs).

iScore Calculation:

The iScore is a multi-parameter calculation provided within the iTopiasoftware. Its value was calculated based on the binding, affinity andstability data.

Example 32 Validation of the Antigenicity of PSMA₂₈₈₋₂₉₇

HHD transgenic mice (n=4) were immunized with PSMA₂₈₈₋₂₉₇ peptide (25 μgin 25 μl of PBS, plus 12.5 μg of pI:C to each lymph node) at day 0, 3,14 and 17. One week after the boost, splenocytes were stimulated ex vivowith the native PSMA₂₈₈₋₂₉₇ peptide and tested against ⁵¹Cr-labeledhuman tumor cells (PSMA⁺ A2⁺ LnCap cells, or as negative control, LnCapcells coated with MHC class I-blocking antibody) at various E:T ratios.The results expressed as % specific lysis (mean±SEM), showed thatPSMA-specific T cells were able to lyse human tumor cells in a fashiondependent on MHC class I availability, confirming display of the PSMAepitope on MHC class I of tumor cells, in a fashion allowing immunemediated attack (FIG. 18).

Examples 33-38 Testing of PSMA₂₈₈₋₂₉₇ Analogs

The analogs listed in FIGS. 19 and 20 were tested for various propertiessuch as improved affinity and stability of binding, cross-reactivitywith the native epitope, and immunogenicity as follows in Examples33-38.

Example 33 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Single Position

Using the procedures described in Example 31 the binding characteristicsof PSMA₂₈₈₋₂₉₇ and analogs were assessed in comparison to each other(see FIG. 19). The positive control for binding was melan-A₂₆₋₃₅ A27L.Cross reactivity with the native epitope was assessed by using theanalog peptides to stimulate IFG-gamma secretion from a T cell linesspecific for the native epitope, essentially as described in example 6.The data shown in FIG. 19 was generated by stimulating with 10 μg/ml ofanalog (approximately 10 μM). This concentration generally resulted inmaximal or near-maximal IFN-gamma production for the analogs and thuswas chosen to represent cross-reactivity.

The observed affinities of the analogs are reported in FIG. 19 as ED50s.Met, Ile, Gln, Val, Nva, Nle, and Abu were substituted at the P2position. These generally resulted in similar affinity. The Nle and Metsubstitutions also maintained similar stability of binding, measured ashalf-time of dissociation in hours. The Val, Nva, and Abu analogselicited a similar level of IFN-gamma production.

Val, Leu, Nva, and Nle were substituted for the Ile at the PΩ primaryanchor position. All four had similar binding affinity. The Val and Nvasubstitutions improved the stability of binding and increased the amountof IFN-gamma produced, indicating cross-reactivity and that the analogscan have improved immunogenicity.

The Ser, Sar, and Abu substitutions at P1 maintained similar bindingcharacteristics but had marginally similar cross-reactivity. The Ala,Leu, Ser, and Thr substitutions at the PΩ−1 position also maintainedsimilar binding characteristics. Finally the Trp substitution at P3exhibited affinity and stability of binding that were both increasedabout twofold and IFN-gamma production that was within twofold of thenative peptide, all generally similar values.

Example 34 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Two Positions

The pattern seen above, that substitutions in this epitope did notgreatly impair binding affinity, continued with the double substitutionsexamined (FIG. 20) which uniformly displayed similar or improved bindingaffinity compared to the native peptide. Among the analogs withsubstitutions at both primary anchor positions those with Nva of Nle atP2 and Val at PΩ, and Val at P2 and Nva at PΩ displayed improved bindingstability and the former two increased IFN-gamma production (data notavailable for the 3^(rd) analog). The Val and Nva substitutions at PΩwere also paired with Ala and Abu substitutions at P1. These analogs allhad robust binding stability and IFN-gamma production that was improvedcompared to the single PΩ substitutions, thus further improving the P1substitutions. The PΩ Nva substitution was also able to restore similarcross-reactivity to the P3 Trp substitution.

Example 35 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Three Positions

Triple substitutions as P1, P2, and P3; P1, P2, and PΩ; P2, P3, and PΩ;and P1, P3, and PΩ were made (FIG. 21). In all cases the P1 substitutionwas Ala, the P3 substitution was Tip, and the PΩ substitution Val orNva. As above affinity at least similar to the native peptide wasmaintained. For the P1, P2, P3 class Nva and Nle at P2 improved thestability of binding. This P2 Nva analog elicited a similar amount ofIFN-gamma while the Nle analog showed a substantial increase.

For the P1, P2, PΩ class, Nva and Val at P2 and PΩ in either combinationimproved binding stability. This P2 Nva PΩ Val analog also showed asubstantial increase in IFN-gamma production. (No data on the other).Val at both P2 and PΩ in this triple substitution showed bindingstability and IFN-gamma production that was nearly halved from that ofthe native peptide.

For the P2, P3, PΩ group only the Nva/W/V analog showed improved bindingor IFN-gamma production. For the two P1, P3, PΩ analogs examined PΩ ofVal or Nva improved binding stability but poor cross-reactivity.

Example 36 Cross-Reactive Immunogenicity of Various Analogs

Groups of HHD transgenic mice (n=8) were immunized with peptide

(natural epitope PSMA₂₈₈₋₂₉₇, or analogs bearing substitutions atprimary or secondary anchor residues), by direct inoculation into theinguinal lymph nodes, with 25 μg in 25 μl of PBS+12.5 μg of pI:C to eachlymph node at day 0, 3, 14 and 17.

Mice were sacrificed at 10 days after the last boost, splenocytesprepared and assessed for IFN-γ production by ELISPOT analysis. Variousnumbers of splenocytes/well were stimulated with 10 μg/ml of nativepeptide in ELISPOT plates coated with anti-IFN-γ antibody. At 48 hoursafter incubation, the assay was developed and the frequency ofcytokine-producing T cells that recognized native PSMA₂₈₈₋₂₉₇ peptidewas automatically counted. The data were represented in FIG. 22 as thenumber of spot forming colonies/well (mean of triplicates±SD). The datashow increased priming of immune responses against the native epitopeachieved by the I297V and P290W analogs, with the other analogs showingslightly higher (but significant) activity than the native peptide(I297Nva or G288Abu or L289Nle I297Nva). To the extent that the poorimmunogenicity of the native epitope reflects tolerance, the improvedactivity of these analogs represents tolerance breaking.

Example 37 Amplification by the I297V Analog of the Response toPSMA₂₈₈₋₂₉₇ Induced by Plasmid

Two groups of HHD transgenic mice (n=8) were immunized with plasmidexpressing PSMA₂₈₈₋₂₉₇, by direct inoculation into the inguinal lymphnodes with 25 μg in 25 μl of PBS to each lymph node at day 0, 3, 14 and17. This was followed by two peptide boosts (25n) at day 28 and 31 witheither the natural peptide or the I297V analog.

Mice were sacrificed at 10 days after the last boost, splenocytesprepared and assessed for IFN-γ production by ELISPOT analysis. Variousnumbers of splenocytes/well were stimulated with 10 ug/ml of nativepeptide in ELISPOT plates coated with anti-IFN-γ antibody. At 48 hoursafter incubation, the assay was developed and the frequency ofcytokine-producing T cells that recognized the PSMA₂₈₈₋₂₉₇ peptide wasautomatically counted. The data were represented in FIG. 23 as frequencyof specific T cells normalized to 0.5 million responder cells (mean oftriplicates+SD). The data show that irrespective of the number ofsplenocytes/well, the frequency of native epitope-specific T cells wasconsiderably higher in the mouse group immunized with the I297V analog.

Example 38 Ex Vivo Cytotoxicity Against Human Tumor Cells

HHD transgenic mice (n=4) were immunized with plasmid expressing thePSMA₂₈₈₋₂₉₇ epitope, by direct inoculation into the inguinal lymph nodeswith 25 μg in 25 μl of PBS to each lymph node at day 0, 3, 14 and 17.This was followed by two peptide boosts (same amount) at day 28 and 31,with the analog I297V. One week after the boost, splenocytes werestimulated ex vivo with the native PSMA₂₈₈₋₂₉₇ peptide and testedovernight against ⁵¹Cr-labeled human tumor cells (Lncap, A2⁺ PSMA⁺; or624.38 A2⁺ PSMA⁻ or control 624.28 cells A2⁻ PSMA⁻) at various E:Tratios. The resulting immunity was effective in mediating cytotoxicityagainst Lncap (FIG. 24).

Example 39 Validation of the Antigenicity of PRAME₄₂₅₋₄₃₃

HHD transgenic mice (n=4) were immunized with PRAME₄₂₅₋₄₃₃ peptide (25μg in 25 μl of PBS, plus 12.5 μg of pI:C to each lymph node) at day 0,3, 14 and 17. One week after the boost, splenocytes were stimulated exvivo with the native PRAME₄₂₅₋₄₃₃ peptide and tested against⁵¹Cr-labeled human tumor cells (PRAME⁺ A2⁺ 624.38 melanoma cells; ornegative control 624.38 cells, deficient in A2 expression) at variousE:T ratios. The results expressed as % specific lysis (mean±SEM), showedthat PRAME-specific T cells were able to lyse human tumor cells,confirming display of the PRAME₄₂₅₋₄₃₃ epitope on MHC class I of tumorcells, in a fashion allowing immune mediated attack (FIG. 25).

Examples 40-48 Testing of PRAME₄₂₅₋₄₃₃ Analogs

The analogs listed in FIGS. 26-28 were tested for various propertiessuch as improved affinity and stability of binding, cross-reactivitywith the native epitope, and immunogenicity as follows in Examples40-48. Using the procedures described in Example 31 the HLA-A*0201binding characteristics of PRAME₄₂₅₋₄₃₃ and 69 analogs were assessed incomparison to each other. The positive control for binding wasmelan-A₂₆₋₃₅ A27L. The observed affinities of the analogs are reportedas % binding (compared to the positive control) and ED50s. Stability ofbinding as half time of dissociation. Cross reactivity with the nativeepitope was assessed by using the analog peptides to stimulate IFG-gammasecretion from a T cell lines specific for the native epitope,essentially as described in Example 6. The data shown in FIGS. 26-28were generated by stimulating with analog peptide at approximately0.304. The results were collected from three separate experiments andwere normalized to the amount of IFN-γ elicited by the native peptide ineach. In some cases the reported values are the average of twodeterminations. An asterisk “*” indicates that IFN-γ production was notdistinguishable from background.

Example 40 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at a Single Position (FIG. 26)

Single substitutions of Val, Met, Ile, Nle, Nva, and Abu were made forthe Leu at the P2 primary anchor position. All of these analogsexhibited % binding within 20% of the native peptide. The ED50 wasdetermined for the Met and Nva analogs. The former had an affinitysomewhat improved but comparable to the native peptide while thelatter's affinity was reduced about 3-fold, but was still comparable tothe PSMA₂₈₈₋₂₈₇ epitope. All of the P2 substitutions maintained bindingstability at least similar to the native peptide. The Met, Nle, and Nvaanalogs elicited IFN-γ production within twofold of the native peptideand the Val analog somewhat less.

Single substitution of Lys, Phe, Tyr, Thr, Orn (ornithine), and Hse(homoserine) were made for the Ser at the P1 position. All of theseanalogs exhibited % binding within 20% of the native peptide except forthe Phe analog which exceeded that range on the high side. The ED50 forthe Lys analog has not been determined, but the other five analogs hadaffinities within threefold of the native peptide. Stability of bindingwas generally similar to the native peptide with the Phe P1 analogshowing greatest binding stability in this group with a half time ofdissociation of 17.7 hours compared to 12.2 hours for the nativepeptide. With the exception of the Lys P1 analog, which elicited 40% ofthe IFN-γ of the native peptide, all of these analogs were consideredcross-reactive as they elicited IFN-γ production within twofold of thenative peptide.

Single substitutions of Val, Ile, Ala, Nle, Nva, Abu, were made to thePΩ anchor position, as well as modifying the carboxy-terminus by theaddition of an amide group. Measured binding affinities were at leastsimilar to native peptide. Stability of binding was also generallysimilar with outliers of the Nva analog at the high end, t1/2 of 17.2hours, and the C-terminal amide at the low end with a significantlyreduced t1/2 of only 3 hours. The Val, Ile, Ala, and Abu PΩ analogsexhibit less preferred cross-reactivity, but the others elicited IFN-γproduction within twofold of the native peptide.

Single substitutions at positions primarily affecting TCR interactionswere also made: Nle, Nva, and Abu at P3 and P6, and Ala, Ser, and Sar atP8. The P6 Nva analog produced IFN-γ within twofold of that of thenative peptide, though the P6 Abu analog was close at 44%.

Example 41 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Two Positions

Double substitution analogs were created at P1 and P2, P2 and PΩ, and P1and PΩ using various combinations of the single substitutions above(FIGS. 27A and 27B). None of the P 1-P2 double substitutions examinedhad radical changes to binding affinity or stability, but none of theexhibited significant cross-reactivity in the IFN-γ assay. A similarpattern is seen with the P2-1²12 double substitution analogs, however,the L426Nva L433Nle analog shows a significant level of cross-reactivitywith the native peptide in the IFN-γ assay along with its similar,somewhat improved binding characteristics. Finally for the P1-PΩ doublesubstitutions, the examined analogs also conformed to the generalpattern of having at least similar binding characteristics, buteliciting negligible IFN-γ in the cross-reactivity assay. The exceptionin this grouping were the S425T L433Nle analog, which exhibited somewhatimproved binding stability and significant cross-reactivity, and theS425F L433Nle analog, which had a more that fourfold reduced ED₅₀, anearly doubled halftime of dissociation, and elicited more IFN-γ thanthe native peptide.

Example 42 Cross-Reactivity and Functional Avidity of AnalogsSubstituted at Three Positions

Four triple substitution analogs were investigated, having Phe or Thr atP1, Nva or Met at P2, and Nle at PΩ. The S425T L426M L433Nle analog hadsimilar affinity whereas the affinity was improved for the other threeanalogs. Both analogs with P2 Nva substitutions displayed increasedstability of binding and significant levels of cross-reactivity. SeeFIG. 28.

Example 43 Cross-Reactive Immunogenicity of the L426Nva L433Nle Analog

Two groups of HHD transgenic mice (n=8) were immunized with a plasmid,pCTLR2 described in example 49 below, expressing PRAME₄₂₅₋₄₃₃ by directinoculation into the inguinal lymph nodes of 25 μg in 25 μl of PBS toeach lymph node at day 0, 3, 14 and 17. This was followed by two peptideboosts (2.5 μg) at day 28 and 31, of native peptide or the PRAME epitopeanalog L426Nva L433Nle.

Mice were sacrificed at 10 days after the last boost, splenocytesprepared and assessed for IFN-γ production after in vitro stimulation at0.5×10⁶ cells/well, with 10 ug/ml of native peptide. At 48 hours afterincubation, the supernatant was harvested and the concentration of IFN-γproduced in response to the PRAME₄₂₅₋₄₃₃ peptide was measured by ELISA.The data were represented in FIG. 29 and show a significant enhancementof IFN-γ production in mice boosted with the PRAME₄₂₅₋₄₃₃ L426NvaL433Nle analog.

Example 44 In Vivo Cytotoxicity Induced by the L426Nva L433Nle Analog

Two groups of HHD transgenic mice (n=8) were immunized as described inExample 43 above.

To evaluate the in vivo responses obtained against the native epitope,splenocytes were isolated from littermate control HHD mice and incubatedwith 20 μg/mL or 1 μg/ml of native peptide for 2 hours. These cells werethen stained with CFSE^(hi) and CFSE^(med) fluorescence (4.0 μM or 1 μM,respectively, for 15 minutes) and intravenously co-injected intoimmunized mice with an equal number of control splenocytes stained withCFSE^(lo) fluorescence (0.4 μM). Eighteen hours later the specificelimination of target cells was measured by removing the spleens andPBMC from challenged animals and measuring CFSE fluorescence by flowcytometry. The relative depletion of the populations corresponding topeptide loaded splenocytes was calculated relative to the control(unloaded) population and expressed as % specific lysis. The results inFIG. 30 showed preserved induction of cytotoxicity when the analogreplaced the natural peptide as a booster agent. The trend indicatesthat the analog can improve on induction of cytotoxic immunity.

Example 45 In Vivo Cytotoxicity and Tetramer Staining

Seven groups of HHD transgenic mice (n=4) were immunized with a plasmid,pCTLR2, expressing PRAME₄₂₅₋₄₃₃ by direct inoculation into the inguinallymph nodes of 25 μg in 25 μl of PBS to each lymph node at day 0, 3, 14and 17. This was followed by two peptide boosts (2.5 μg) at day 28 and31, of native peptide, negative control (EAAGIGILTV (SEQ ID NO. 145)peptide), or PRAME₄₂₅₋₄₃₃ epitope analogs bearing mutations at theprimary and/or secondary anchor residues—S425F, L426Nva L433Nle, S425TL433Nle, and S425T L426Nva L433Nle.

To evaluate the in vivo response against native peptide, splenocyteswere isolated from littermate control HHD mice and incubated with 0.2ug/ml or 20 ug/ml of native peptide for 2 hours. These cells were thenstained with CFSE fluorescence (1 and 2.5 μM respectively, for 15minutes) and intravenously co-injected into immunized mice with an equalnumber of control splenocytes stained with CFSE^(lo) fluorescence (0.4μM). Eighteen hours later the specific elimination of target cells wasmeasured by removing the spleen from challenged animals and measuringCFSE fluorescence in the resulting cell suspensions, by flow cytometry.The relative depletion of the populations corresponding topeptide-loaded splenocytes was calculated relative to the control(unloaded) population and expressed as % specific lysis. In addition,the frequency of PRAME₄₂₅₋₄₃₃-specific T cells, was evaluated bytetramer/CD8 co-staining The boost with analogs encompassing mutationsat primary or secondary anchor residues showed comparable immuneactivity as compared to the native peptide, based on in vivocytotoxicity and tetramer staining The analogs were capable ofamplifying the immune response as shown by comparison with the “EAA”group, boosted with an irrelevant peptide. In that regard, analogscomprising S425F, L33Nle, L426Nva L433Nle, S425T L433Nle, or S425TL426Nva L433Nle were all capable of expanding the immunity against thenative epitope, as assessed by in vivo cytotoxicity. However, only theL433Nle, L426Nva L433Nle, and S425T L426Nva L433Nle analogs expanded thesubset of T cells specific against the native epitope to a levelsignificantly higher that in mice primed with plasmid and boosted withthe negative control peptide (FIG. 31).

Example 46 Ex Vivo Cytokine Production

Three groups of HHD transgenic mice (n=4) were immunized with a plasmid,pCTLR2, expressing PRAME₄₂₅₋₄₃₃ by direct inoculation into the inguinallymph nodes of 25 μg in 25 μl of PBS to each lymph node at day 0, 3, 14and 17. This was followed by two peptide boosts (2.5 μg) at day 28 and31, of the PRAME epitope analogs L426Nva L433Nle and S425T L426NvaL433Nle or the negative control peptide Melan A (EAAGIGILTV (SEQ ID NO.145)).

Mice were sacrificed at 10 days after the last boost, splenocytesprepared and assessed for IFN-γ production by ELISA at 48 hours afterincubation with 10 μg/ml of native peptide. The data were represented inFIG. 32 as cytokine concentration in pg/ml (mean of triplicates±SD). Thedata showed ex vivo cytokine production by splenocytes from mice boostedwith both analogs, and greater response to L426Nva L433Nle than to S425TL426Nva L433Nle.

Example 47 Ex Vivo Cytotoxicity Against a Human Tumor Cell Line afterPeptide Boost with Analog

HHD transgenic mice (n=4) were immunized with a plasmid, pCTLR2,expressing PRAME₄₂₅₋₄₃₃ by direct inoculation into the inguinal lymphnodes of 25 μg in 25 μl of PBS to each lymph node at day 0, 3, 14 and17. This was followed by two peptide boosts (2.5 μg) at day 28 and 31,with the analog L426Nva L433Nle. One week after the boost, splenocyteswere stimulated ex vivo with the native peptide and tested against⁵¹Cr-labeled human tumor cells (PRAME⁺ 624.38 melanoma cells pretreatedor not with IFN-γ; or negative control 624.38 cells, deficient in HLA-A2expression) at various E:T ratios. The analog L426Nva L433Nle elicitedimmune responses that mediated significant cytotoxicity against humantumor cells expressing A2 (624.38), slightly elevated upon theirpre-treatment with IFNγ. In contrast, no significant activity wasmeasured against A2-624.28 control cells. See FIG. 33.

Example 48 In Vitro Immunization to PRAME₄₂₅₋₄₃₃

In vitro immunization was carried out according to the general schemepresented in FIG. 34. Peripheral blood mononuclear cells (PBMCs) wereobtained from healthy donors (HLA-A*0201⁺) by Ficoll-separation. FreshPBMCs (2.5×10⁶), together with 5 ng/ml PRAME₄₂₅₋₄₃₃ or peptide analogwere plated in T-cell culture medium. Subsequently 20 IU/ml ofinterleukin 2 was added to each well after 72 and 96 hours and additionpeptide (5 ng/ml) was added at day 7. Cultures were maintained for anadditional 10 days before effector cells were harvested and used intetramer staining IVS PBMCs were labeled with PRAME₄₂₅₋₄₃₃ tetramer andanalyzed on the FACSCalibur (BD, San Jose, Calif.). Quadrants were setbased on negative controls, stained with irrelevant HBV tetramer andSSX2 tetramer, and a minimum of 10,000 gated events were captured.Tetramer-positive cells are expressed as a percentage of the lymphocytepopulation. PRAME₄₂₅₋₄₃₃-specific tetramers was significantly enhancedfollowing IVS with peptide analog as compared with native peptide. SeeFIG. 35. This demonstrates that the analog can be a preferableimmunogen.

Example 49 pCTLR2, a Plasmid Expressing the PRAME₄₂₅₋₄₃₃ Epitope

pCTLR2 is a recombinant DNA plasmid vaccine which encodes onepolypeptide with an HLA A2-specific CTL epitope, SLLQHLIGL (SEQ ID NO.115), from PRAME amino acid residues 425-433, and an epitope clusterregion of PRAME, amino acids 422-509. The cDNA sequence for thepolypeptide in the plasmid is under the control of promoter/enhancersequence from cytomegalovirus (CMVp) which allows efficienttranscription of messenger for the polypeptide upon uptake by antigenpresenting cells. The bovine growth hormone polyadenylation signal (BGHpolyA) at the 3′ end of the encoding sequence provides signal forpolyadenylation of the messenger to increase its stability as well astranslocation out of nucleus into the cytoplasm. To facilitate plasmidtransport into the nucleus, a nuclear import sequence (NIS) from Simianvirus 40 has been inserted in the plasmid backbone. One copy of CpGimmunostimulatory motif is engineered into the plasmid to further boostimmune responses. Lastly, two prokaryotic genetic elements in theplasmid are responsible for amplification in E. coli, the kanamycinresistance gene (Kan R) and the pMB bacterial origin of replication.(See FIG. 36).

Immunogen Translation Product Sequence

The amino acid sequence of the encoded polypeptide (150 amino acidresidues in length) is given below.

(SEQ ID NO. 147) MALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPNKRSLLQHLIGLGDAAYSLLQHLIGLISPEKEEQYIA SLLQHLIGLKRPSIKRSLLQH LIGL.

The first 89 amino acid residues are an epitope cluster regionrepresenting PRAME 422-509. Within this epitope cluster region, a numberof potential HLA A2-specific CTL epitopes have been found using avariety of epitope prediction algorithms. Amino acid residues 90-150 arean epitope liberation (SYNCHROTOPE™) sequence with four copies of PRAME425-433 CTL epitope (boldface) embedded. Flanking the defined PRAME CTLepitope are short amino acid sequences that have been shown to play animportant role in the processing of the PRAME CTL epitope. In addition,the amino acid sequence ISPEKEEQYIA (SEQ ID NO. 150) (corresponding toPRAME amino acid 276-286, in italics) is engineered into thesting-of-beads region to facilitate the detection of expression ofencoded polypeptide.

Using a variety of immunological assays including tetramer, ELISPOT,ELISA, and cytotoxicity, strong CTL responses specific for epitopePRAME₄₂₅₋₄₃₃ have been detected from HLA-A2 transgenic mice immunizedwith the pCTLR2 plasmid, suggesting immunogenic potency for pCTLR2.These data indicated that the plasmid has been taken up by antigenpresenting cells, the encoded polypeptide has been synthesized andproteolytically processed to produce the nonamer epitope peptide, andbecome HLA-A2 bound for presentation.

Plasmid Construction

Stepwise ligation of sets of long complementary oligonucleotidesresulted in generation of cDNA encoding amino acid residues in the“String-of-Beads” epitope liberation sequence (amino acids 90-150).These cDNA bore appropriate cohesive ends for restriction enzymes thatcan be used for further ligation with cDNA encoding the PRAME epitopecluster region (amino acid 1-89), which were amplified by performing PCRon cDNA encoding PRAME as template. The entire insert was then ligatedinto vector backbone between Afl II and EcoR I sites. The entire codingsequence was verified by DNA sequencing.

Example 50 Generation of Antigen Specific T Cell Responses

H-2 class I-negative, HLA-A2.1-transgenic HHD mice were housed underpathogen-free conditions and used for evaluation of the immunogenicityof HLA-A2.1-restricted human tumor-associated cytotoxic T lymphocyte(CTL) epitopes. Female mice 8-12 weeks of age were used forintralymphatic immunization and for isolation of splenocytes for in vivocytotoxicity studies. The mice were immunized via bilateral inguinallymph node injection. Mice were anesthetized by inhalation ofisofluorane and surgeries were conducted under aseptic conditions.Following preparation for surgery, an incision 0.5 cm in length was madein the inguinal fold and the inguinal lymph node was exposed. A maximumvolume of 25 μl (25 μg) of plasmid DNA vaccine or peptide was injecteddirectly into the lymph node using a 0.5 mL insulin syringe. The woundwas closed with sterile 6-0 nylon skin sutures.

Example 51 Ex Vivo Cytotoxicity Against Human Tumor Cells

HHD transgenic mice (n=4/group) were immunized with the plasmid pSEM(described more fully in U.S. patent application Ser. No. 10/292,413(Pub. No. 20030228634 A1)) incorporated by reference above in itsentirety) expressing melan-A₂₆₋₃₅ A27L epitope analog, by directinoculation into the inguinal lymph nodes with 25 ug in 25 ul ofPBS/each lymph node at day 0, 3, 14 and 17. This was followed by twoadditional peptide boosts (same amount) at day 28 and 31, with theanalogs A27L, A27Nva, or A27L V35Nva. One week after the boost,splenocytes were stimulated ex vivo with the native melan-A₂₆₋₃₅ peptideand tested against ⁵¹Cr-labeled human tumor cells (624.38 cells) atvarious E:T ratios. The resulting immunity after boosting with the A27Lor A27Nva analogs was comparable and more effective than the nativepeptide EAAGIGILTV (FIG. 37). Since the priming plasmid expresses theA27L analog the experiment had a potential bias in favor that peptide,so that the substantial cytotoxicity obtained with the A27Nva analog maybe an underestimate of it potency if priming made use of that samesequence.

Example 52 Tetramer Analysis

Enumeration of CD8+ antigen-specific T cells requires cognaterecognition of the T cell receptor (TCR) by a Class I MHC/peptidecomplex. This can be done using Class I MHC tetramers which are composedof a complex of four HLA MHC Class I molecules each bound to thespecific peptide and conjugated with a fluorescent protein. Thustetramer assays allow quantitation of the total T cell populationspecific for a given peptide complexed in a particular MHC molecule.Furthermore, since binding does not depend on functional pathways, thispopulation includes all specific CD8+ T cells regardless of functionalstatus. The CTL response in immunized animals was measured byco-staining mononuclear cells isolated from peripheral blood afterdensity centrifugation (Lympholyte Mammal, Cedarlane Labs) withHLA-A*0201 MART1 (ELAGIGILTV)-PE MHC tetramer (Beckman Coulter, T01008)or a Tyrosinase₃₆₉₋₃₇₇ (YMDGTMSQV) specific tetramer reagent (HLA-A*0201Tyrosinase-PE, Beckman Coulter) and FITC conjugated rat anti-mouse CD8a(Ly-2) monoclonal antibody (BD Biosciences). Data was collected using aBD FACS Calibur flow cytometer and analysed using cellquest software bygating on the lymphocyte population and calculating the percent oftetramer⁺ cells within the CD8⁺ CTL population.

Example 53 Tetramer Staining Plasmid Priming, Peptide Boost—NativeVersus Analog

Two groups of HHD transgenic mice (n=8) were immunized with plasmidexpressing Tyrosinase 369-377, by direct inoculation into the inguinallymph nodes with 25 ug in 25 ul of PBS/each lymph node at day 0, 3, 14and 17. This was followed by two additional peptide boosts (similaramount) at day 28 and 31, of natural peptide or the 377Nva analog. Tendays later, the immune response was monitored using a Tyrosinase 369-377specific tetramer reagent (HLA-A*0201 Tyrosinase-PE, Beckman Coulter).Individual mice were bled via the retro-orbital sinus vein and PBMC wereisolated using density centrifugation (Lympholyte Mammal, CedarlaneLabs) at 2000 rpm for 25 minutes. PBMC were co-stained with a mousespecific antibody to CD8 (BD Biosciences) and the Tyrosinase tetramerreagent and specific percentages were determined by flow cytometeryusing a FACS caliber flow cytometer (BD). The percentages of Tyrosinasespecific CD8⁺ cells, show that replacement of the native peptide withthe analog, preserved the expansion of Tyrosinase-specific subset. Thetrend indicates that the analog can improve on the expansion ofTyrosinase specific T cells (FIG. 38).

Example 54 In Vivo Cytotoxicity and Tetramer Staining (Head to HeadComparison Between Native Peptide and a Panel of Analog Candidates)

Four groups of HHD transgenic mice (n=6) were immunized with plasmid(pSEM) expressing Tyrosinase₃₆₉₋₃₇₇ and Melan-A₂₆₋₃₅ A27L epitopes, bydirect inoculation into the inguinal lymph nodes of 25 ug of plasmid in25 ul of PBS per lymph node at day 0, 3, 14 and 17. This was followed bytwo peptide boosts (similar amount) at days 28 and 31, of Melan-A₂₆₋₃₅A27L into the left inguinal lymph node and Tyrosinase₃₆₉₋₃₇₇ analogs,bearing substitutions at the primary and/or secondary anchor residues,into the right lymph node. As controls, mice immunized with plasmid onlyor naïve mice were used.

To evaluate the in vivo response against natural Tyrosinase and Melan Aepitopes, splenocytes were isolated from littermate control HHD mice andincubated separately, with 20 ug/ml of natural peptide (Melan-A₂₆₋₃₅ orTyrosinase₃₆₉₋₃₇₇) for 2 hours in HL-1 serum free medium (Cambrex) at aconcentration of 20×10⁶ cells/mL. These cells were then stained withCFSE (Vybrant CFDA SE cell tracer kit, Molecular Probes) (1 and 2.5 μMrespectively, for 15 minutes) and intravenously co-injected intoimmunized or naïve control HHD mice with an equal number of controlnon-peptide coated splenocytes stained with CFSE^(lo) fluorescence (0.4μM). Eighteen hours later the specific elimination of target cells wasmeasured by removing the spleen from challenged animals and measuringCFSE fluorescence in the resulting cell suspensions, by flow cytometry.The relative depletion of the populations corresponding topeptide-loaded splenocytes was calculated relative to the control(unloaded) population and expressed as % specific lysis. In addition,the frequency of Tyrosinase₃₆₉₋₃₇₇- and Melan-A₂₆₋₃₅-specific T cells,was evaluated by tetramer/CD8 co-staining (HLA-A*0201-tetramers, BeckmanCoulter).

The tyrosinase analog V377Nva was capable of expanding the population oftyrosinase-specific T cells and amplifying cytotoxic immunity, similarlyto the native peptide and greater than the Tyrosinase analog M370VV377Nva (FIG. 39).

Example 55 Ex Vivo Cytotoxicity Against Human Tumor Cells

HHD transgenic mice (n=4/group) were immunized (according to the generalprotocol in FIG. 40) with plasmid (pSEM) expressing theTyrosinase₃₆₉₋₃₇₇ epitope, by direct inoculation into the inguinal lymphnodes of 25 ug of plasmid in 25 ul of PBS per lymph node at day 0, 3, 14and 17. This was followed by two peptide boosts (same amount) at day 28and 31, with the native peptide or analogs bearing substitutions atprimary anchor residues P2 and PΩ (370 and 377). One week after theboost, splenocytes were stimulated ex vivo with the nativeTyrosinase₃₆₉₋₃₇₇ peptide and assayed against ⁵¹Cr-labeled human tumorcells (624.38 cells) at various E:T ratios. Both the native peptide andthe M370V V377Nva analog generated robust cytotoxicity against 624.38cells (FIG. 41). Whereas there was some dilution of cytolytic activitywith the native peptide there was none with the analog reinforcing theindication of greater immunogenicity gained from the tetramer results inExample 52. Together with the preceding example, this observationillustrates the usefulness of complementing more stringent assays (invivo cytotoxicity and tetramer staining) with more sensitive assays (exvivo cytotoxicity after in vitro stimulation), to outline potentiallyuseful analogs.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods may beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein.

Furthermore, the skilled artisan will recognize the interchangeabilityof various features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the invention extends beyond the specifically disclosedembodiments to other alternative embodiments and/or uses and obviousmodifications and equivalents thereof.

What is claimed is:
 1. An isolated peptide comprising one to threesubstitutions in the sequence KASEKIFYV (SEQ ID NO. 1), wherein the oneto three substitutions are at amino acid position 1, 2, 4, 6, 8, or 9,wherein the peptide has an affinity for a class I MHC binding cleft thatis similar to or greater than the affinity of KASEKIFYV (SEQ ID NO. 1)for said class I MHC binding cleft, wherein the one to threesubstitutions at amino acid position 1, 2, 4, 6, 8, or 9 are selectedfrom the group consisting of: a substitution at position 1 with anaromatic amino acid or D-Lys, a substitution at position 2 with ahydrophobic amino acid and/or an amino acid with a bulky side chain, asubstitution at position 4 with a polar amino acid, a substitution atpositions 6 with a polar amino acid, or an amino acid with a largealiphatic side chain, a substitution at position 8 with a polar aminoacid or an aromatic amino acid, and a substitution at position 9 with alarge aliphatic amino acid, and wherein said isolated peptide is not apeptide of the sequence K{D-Ala}SEKIFYV or KASEKIFY{V—NH2}.
 2. Theisolated peptide of claim 1 wherein the halftime of dissociation issimilar to or greater than the halftime of dissociation of KASEKIFYV(SEQ ID NO. 1) from said class I MHC binding cleft.
 3. The isolatedpeptide of claim 1 that is recognized by T cells with specificity forthe peptide KASEKIFYV (SEQ ID NO. 1).
 4. The isolated peptide of claim1, wherein the peptide has affinity for a class I MHC peptide bindingcleft.
 5. The isolated peptide of claim 4, wherein the class I MHC isHLA-A2.
 6. A class I MHC/peptide complex wherein the peptide has thesequence of the peptide of claim 1 and is complexed with a MHC protein.7. The class I MHC/peptide complex of claim 6, that is cross-reactivewith a TCR that recognizes a class I MHC/SSX-2₄₁₋₄₉ complex.
 8. Theclass I MHC/peptide complex of claim 7, wherein the class IMHC/SSX-2₄₁₋₄₉ complex is an HLA-A2/SSX-2₄₁₋₄₉ complex.
 9. A polypeptidecomprising the peptide sequence of claim 1, in association with aliberation sequence.
 10. An immunogenic composition comprising thepeptide of claim
 1. 11. An immunogenic composition comprising thepolypeptide of claim
 9. 12. Nucleic acid means for expressing thepeptide of claim
 1. 13. A nucleic acid encoding the polypeptide of claim9.
 14. An immunogenic composition comprising the nucleic acid of claim12.
 15. An immunogenic composition comprising the nucleic acid of claim13.
 16. A method of inducing, maintaining, entraining, or amplifying aCTL response comprising intranodal administration of the composition ofclaim
 11. 17. A method of inducing, maintaining, entraining, oramplifying a CTL response comprising intranodal administration of thecomposition of claim
 10. 18. The method of claim 17, wherein the T cellresponse recognizes the native peptide.
 19. A method of inducing,maintaining, entraining, or amplifying a class I MHC-restricted T cellresponse comprising intranodal administration of the composition ofclaim 11 plus an immunopotentiating agent.
 20. A method of inducing,maintaining, or entraining a CTL response comprising intranodaladministration of the composition of claim 14.